Compositions and methods for analyte detection

ABSTRACT

The inventions provided herein relate to detection reagents, compositions, methods, and kits comprising the detection reagents for use in detection, identification, and/or quantification of analytes in a sample. Such detection reagents and methods described herein allow multiplexing of many more labeled species in the same procedure than conventional methods, in which multiplexing is limited by the number of available and practically usable colors.

RELATED APPLICATIONS

This application is a continuation application which claims priority toU.S. patent application Ser. No. 16/255,920, filed on Jan. 24, 2019,which is a continuation application which claims priority to U.S. patentapplication Ser. No. 14/366,486, filed on Jun. 18, 2014, which is aNational Stage Application under 35 U.S.C. 371 of co-pending PCTApplication No. PCT/US12/71398 designating the United States and filedDec. 21, 2012; which claims the benefit of U.S. Provisional ApplicationNo. 61/579,265 and filed Dec. 22, 2011 each of which are herebyincorporated by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under HG005550 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD OF THE DISCLOSURE

The inventions provided herein relate to detection molecules ordetection reagents, compositions, methods and kits for detection,identification, and/or quantification of analytes in a sample.

BACKGROUND

The need for multiplexing techniques in biology is often driven by thefact that test samples are precious and those analyzing them either donot know in advance precisely what to look for or must extract the mostinformation from any single sample. Hence, it is desirable forclinicians and researcher to subject each sample to a large set ofprobes.

Optical readout is common in biology and can be very effective. However,it is typically limited to a relatively small number of availablefluorophores or chromophores (which are referred to collectively ascolors). In practice, multiplexing by fluorescence is often limited to 4or 5 colors, which by traditional methods implies that at most 4 or 5probes can be detected in a single sample.

The common approach to improving multiplexing in optical methods is toincrease the number of available colors. To this end, quantum dots havebeen developed to provide a larger range of colors. However, in reality,it is difficult to use more than 6 quantum dot colors simultaneously.Another approach is to use mixtures or ratio of fluorophores as newcolors. Such methods have extended multiplexing to hundreds of analytes,but due to the size of the labels (e.g., microbeads), the technology hasthus far been limited to flow-cytometry based analyses. Yet anotherapproach involves nanostrings, which are essentially short strings ofstrung-up fluorophores creating visible colorful barcodes.Unfortunately, nanostring readout requires very high-resolution imagingand a special flow apparatus. Further, the nanostrings can only be usedin a sample where the probes' targets are sparse, or the barcodes willoverlap and create a blur.

A simple workaround for the limited number of colors (e.g., 4 or 5colors) in optical readouts is to repeat the probing of the same samplewith multiple small sets of different probes. For example, the assay caninvolve probing the sample with 4 different antibodies at a time andimaging after every assay. If the test requires probing the sample witha total of 64 antibodies, the 4-probe procedure would have to berepeated 16 times using the sample. As such, the order of detectingdifferent target analytes in a single sample may need to be prioritized,because some target analytes in the sample can degrade during successiveprobings. Accordingly, there is still a strong need for accurate andsensitive methods with a high throughput for detection, identification,and/or quantification of target molecules in a sample, e.g., complexmixtures.

SUMMARY

Embodiments provided herein are based on, at least in part, thedevelopment of a multiplexed biological assay and readout, in which amultitude of detection reagents comprising one or more probes and/orprobe types are applied to a sample, allowing the detection reagents tobind target molecules or analytes, which can then be opticallyidentified in a temporally-sequential manner. In some embodiments, themultitude of detection reagents comprising one or more probes and/orprobe types can be applied to a sample simultanesouly. Accordingly,provided herein are methods, compositions (e.g., detection reagents) andkits for detecting multiple analytes in a sample.

Accordingly, one aspect provided herein relates to a method fordetecting a plurality of analytes in a sample. Exemplary analytesinclude, without limitations, antigens, receptors, proteins, peptides,nucleic acids, sugars, lipid, carbohydrates, glycans, glycoproteins,oligonucleotides, cells, viruses, and any combinations thereof. In someembodiments, the nucleic acids can include, e.g., cellular DNA or RNA,messenger RNA, microRNA, ribosomal RNA, and any combinations thereof. Asample amenable to the methods described herein can be a sample from anysources, e.g., but not limited to biological samples, e.g., collectedfrom organisms, animals or subjects, environmental samples, food, foodbyproduct, soil, archaeological samples, extraterrestrial samples, orany combinations thereof. For example, a sample can be a protein sampleimmobilized on a solid support including, e.g., a blotting membrane. Inalternative embodiments, a sample can comprise one or more cells, one ormore tissues, one or more fluids, or any combinations thereof. In someembodiments, the sample can comprise a tissue sample. In someembodiments, the sample can comprise a fluid sample. In someembodiments, a sample can comprise blood, sputum, cerebrospinal fluid,urine, saliva, sperm, sweat, mucus, nasal discharge, vaginal fluids orany combinations thereof. In some embodiments, a sample can comprise abiopsy, a surgically removed tissue, a swap, or any combinationsthereof.

The method described herein comprises: (a) contacting the sample with aplurality of detection reagents as described herein, wherein eachsubpopulation of the detection reagents can target at least onedifferent analyte; and (b) detecting in a temporally-sequential mannersaid plurality of the pre-determined subsequences of said detectionreagents, wherein said detection of the subsequences each generates asignal signature corresponding to said subsequence, and wherein atemporal order of the signal signatures corresponding to said pluralityof the subsequences of said detection reagent identifies a subpopulationof the detection reagents. In some embodiments, the temporal order ofthe signal signatures corresponding to said plurality of thesubsequences of said detection reagent can be unique for eachsubpopulation of the detection reagents.

In some embodiments, a detection reagent described herein can target atleast two distinct analytes. In some embodiments, a first subpopulationof the detection reagents can target at least one analyte different fromthat of a second subpopulation of the detection reagents. Accordingly,in some embodiments, the readout of the detection reagents can bedistinct but overlapping.

In some embodiments, the method can further comprise processing thesample before contacting with the plurality of detection reagentsdescribed herein.

In some embodiments, the method can further comprise removing anyunbound detection reagents before detection of the pre-determinedsubsequences in a temporally-sequential manner.

In some embodiments, the method can further comprise comparing thetemporal order of the signal signatures with different identifiers ofsaid at least one probe reagent, wherein an agreement between thetemporal order of the signal signatures and a particular identifier ofsaid at least one probe reagent identifies the analyte in the sample. Insome embodiments, the method can further comprise measuring theintensity of the signal signatures generated from each subpopulation ofthe detection reagents. In some embodiments, the intensity of the signalsignatures generated from each subpopulation of the detection reagentscan indicate an amount of the analyte. In some embodiments, the relativeintensity of the signal signatures can be used in identification of eachsubpopulation of the detection reagents. Thus, the intensity of thesignal signatures can be used as part of a coding scheme of thedetection reagents described herein. The comparing and intensitymeasuring steps can be performed by a computer-implemented software oralgorithm.

Each signal signature corresponding to individual pre-determinedsubsequences of the detection reagent are detected in atemporally-sequential manner. In some embodiments, the detection methodcan include sequencing, e.g., which can be performed via any methodsknown in the art, including but not limited to, ligation, hybridization,synthesis, amplification, single-base extension, or any methods known inthe art. In certain embodiments, the detection method can includehybridizing a decoder probe with the corresponding subsequence, whereinthe decoder probe can comprise a detectable label.

In particular embodiments, the detection method can comprise: (a)hybridizing a set of decoder probes with a subsequence of the detectionreagents, wherein each subpopulation of the decoder probes can comprisea detectable label, each detectable label producing a signal signature;(b) detecting said different signal signature produced by thehybridization of said set of decoder probes; (c) optionally removingsaid different signal signature produced by the hybridization of saidset of decoder probes; and (d) repeating steps (a) through (c) for othersubsequences of said detection reagents, thereby producing a temporalorder of the signal signatures corresponding to said each detectionreagent. In some embodiments, removal of the different signal signatureproduced by the hybridization can be performed by washing, heating,photo-bleaching, displacement (e.g., displacement of decoder probes withanother reagent or nucleic acid sequence), cleavage, enzymaticdigestion, quenching, chemical degradation, bleaching, oxidation, or anycombinations thereof.

In some embodiments, each decoder probe in the set can independentlyhave a subsequence of the detection reagents.

In some embodiments, each subpopulation of the decoder probes cancomprise a different detectable label, each different detectable labelproducing a different signal signature.

In some embodiments, each subpopulation of the decoder probes can becomplementary (e.g., partially complementary or completelycomplementary) to the subsequence of the detection reagents. In someembodiments, a first subpopulation and a second subpopulation of thedecoder probes can be complementary (e.g., partially complementary orcompletely complementary) to the same subsequence of the detectionreagents. In some embodiments, a first subpopulation and a secondsubpopulation of the decoder probes can be complementary (e.g.,partially complementary or completely complementary) to distinctsubsequences of the detection reagents.

In some embodiments involving decoder probes for detection purposes, thedetectable label associated with each subpopulation of the decoderprobes can comprise an optical label selected from the group consistingof a small-molecule dye, a fluorescent molecule or protein, a quantumdot, a colorimetric reagent, a chromogenic molecule or protein, a Ramanlabel, a chromophore, and any combinations thereof. In some embodiments,the detectable label or optical label can be a fluorescent molecule orprotein.

Types of signal signature(s) can vary upon different embodiments ofdetection reagents and/or decoder probes described herein. By way ofexample, the detection reagents and/or decoder probes can comprise anoptical molecule or label, thus producing optical signatures. Examplesof optical signatures can include, without limitations, signatures offluorescent color (e.g., emission spectra under one or more excitationspectra), visible light, no-color or no-light, color (e.g., colordefined by a visible light wavelength), Raman signatures, and anycombinations thereof. In some embodiments, an optical signature cancomprise signatures of one or more fluorescent colors, one or morevisible lights, one or more no-colors or no-lights, one or more colors,one or more Raman signatures, or any combinations thereof. For example,in one embodiment, an optical signature can comprise a plurality (e.g.,at least 2 or more) of fluorescent colors (e.g., fluorescent dyes). Inthese embodiments, the optical signatures can be detected by opticalimaging or spectroscopy.

The spatial movement limit of an analyte in a sample allowed for atemporal detection of the detection reagents to occur can vary dependingon a number of factors, including, but not limited to, presence of anydistinguishable features within a field of detection, magnification usedin detection (e.g., magnification of the microscope lens), density ofthe analytes in a sample, and any combinations thereof. In someembodiments, there can be no limit in the spatial movement of an analytein a sample during a temporal detection of the detection reagents, forexample, provided that the analyte stay within the field of detectionand there is at least one same distinguishable feature in each imagetaken during a temporal detection so that the images can be aligned toeach other based on the same distinguishable feature. In someembodiments where there is no such distinguishable feature, the spatialmovement of an analyte in a sample can be less than 100 μm, includingless than 50 μm, less than 25 μm, less than 10 μm, less than 1 μm orsmaller, over a time period, during which a temporal detection of thedetection reagents occurs. In some embodiments, the spatial movement ofan analyte in a sample can be less than 1000 nm, including less than 500nm, less than 250 nm, less than 100 nm, less than 50 nm, less than 10 nmor smaller, over a time period, during which a temporal detection of thedetection reagents occurs. More importantly, the spatial movement limitof an analyte in a sample during a temporal detection is determined bythe ability of matching distinguishable features between images takenduring a temporal detection, which can be affected by imagingconditions. In some embodiments, the analyte can be fixed on a solidsubstrate or support.

In one aspect, embodiments provided herein relate to a detectionreagent, which can be used in a multiplexing assay. The detectionreagent comprises at least one probe reagent and at least one nucleicacid label, wherein said at least one nucleic acid label comprises atleast one pre-determined subsequence to be detected in atemporally-sequential manner; wherein said at least one pre-determinedsubsequence forms an identifier of said at least one probe reagent; andwherein said at least one probe reagent and said at least one nucleicacid label are conjugated together.

In some embodiments, the probe reagent and the nucleic acid label can beconjugated together by at least one linker. The linker can be monovalentor multivalent. Exemplary linkers include, but are not limited to, abond, a linker molecule, and/or a particle, for example, selected from agroup consisting of a gold nanoparticle, a magnetic bead ornanoparticle, a polystyrene bead, a nanotube, a nanowire, amicroparticle, and any combinations thereof. In some embodiments, thelinker can be a nanoparticle. Examples of linker molecules can include,but are not limited to, a polymer, sugar, nucleic acid, peptide,protein, hydrocarbon, lipid, polyethelyne glycol, crosslinker, orcombination thereof.

When the linker is a particle, in some embodiments, the particle can bemodified by any methods known in the art. For example, the particle canbe coated with streptavidin or a derivative thereof. In someembodiments, the particles can be modified or functionalized with atleast one functional group. Examples of the functional groups caninclude, but are not limited to, amine, carboxyl, hydroxyl, aldehyde,ketone, tosyl, silanol, chlorine, hydrazine, hydrazide, photoreactivegroups and any combination thereof.

The probe reagent of the detection reagent can be any targeting moleculeof interest. Examples of the probe reagent can include, but are notlimited to, a nucleic acid, an antibody or a portion thereof, anantibody-like molecule, an enzyme, a cell, a virus, an antigen, a smallmolecule, a protein, a peptide, a peptidomimetic, a sugar, a lipid, aglycoprotein, a peptidoglycan, an aptamer, and any combinations thereof.In some embodiments, the probe reagent can be modified by any meansknown to one of ordinary skill in the art. By way of example, the probereagent can be genetically modified, or it can be biotinylated.

The nucleic acid label of the detection reagent can have anyconfiguration and/or any sequence length. In some embodiments, thenucleic acid label can be single-stranded, double-stranded, partiallydouble-stranded, a hairpin, linear, circular, branched, a concatemer(e.g., a concatemer with a 3D structure such as a rolony, i.e.,rolling-circle colony, or a DNA nanoball), or any combinations thereof.In various embodiments, the nucleic acid label can be designed forminimal cross-hybridization of bases with each other.

In some embodiments, the nucleic acid label can be a modified nucleicacid label. An exemplary modification of the nucleic acid labelincludes, without limitations, conjugation of the nucleic acid label toone or more detectable molecules. The detectable molecule can includeany optical molecule, including, but not limited to, a small-moleculedye, a fluorescent protein, a quantum dot, or any combinations thereof.

In some embodiments, the nucleic acid label can comprise at least apartially double-stranded region. For example, the nucleic acid labelcan be pre-hybridized with at least one optically-labeled decoder probe,e.g., to produce at least the first signal of the temporal image stack.In such embodiments, additional decoder probes can be added during thedetection method described herein to hybridize with other single-strandsubsequences of the nucleic acid label.

In some embodiments, the nucleic acid label can comprise a plurality ofpre-determined subsequences. Each of the pre-determined subsequences canbe independently of any length. In some embodiments, at least one of thepre-determined subsequences can comprise one or more nucleobases. Incertain embodiments, at least one of the pre-determined subsequences cancomprise from 1 to 100 nucleobases.

The pre-determined subsequences with the nucleic acid label can beconjugated together by at least one sequence linker. In someembodiments, the sequence linker can be a direct bond, e.g., aphosphoester bond, which can allow conjugation of the pre-determinedsubsequences to form a longer, contiguous sequence. In some embodiments,the sequence linker can be a nucleotidic linker. When the pre-determinedsubsequences are conjugated together by a nucleotidic linker, thenucleotidic linker can have a sequence length of at least onenucleotide. The nucleotidic linker, in some embodiments, can besingle-stranded, double-stranded, partially double-stranded, a hairpin,or any combinations thereof.

Depending on various applications and/or assay conditions (e.g.,sensitivity, sample volume/concentration), a detection signal of a probecan be amplified by conjugating the probe to a plurality of the nucleicacid labels. In such embodiments, the detection reagent can comprise oneprobe reagent and a plurality of nucleic acid labels. Without wishing tobe limiting, in other embodiments, the detection reagent can comprise aplurality of probe reagents and a nucleic acid label. In someembodiments, the detection reagent can comprise a plurality of probereagents and a plurality of nucleic acid labels.

The detection reagents and methods described herein can be used in anybiological assays for detection, identification and/or quantification oftarget molecules or analytes in a sample. In particular embodiments, thedetection reagent can be present in a soluble phase for variousbiological assays. By way of example only, in some embodiments, thedetection reagent can be adapted for use in immunofluorescence. Forexample, the detection reagent adapted for use in immunofluorescence canbe used to identify microbes or pathogens. In alternative embodiments,the detection reagent can be adapted for use in immunohistochemistry.For example, the detection reagent adapted for use inimmunohistochemistry can be used to study tissue biopsies or culturedcells. In some embodiments, the detection reagent and the methoddescribed herein can be applied to fixed cells and/or living cells. Inother embodiments, the detection reagent can be adapted for use influorescence in situ hybridization. In some embodiments, the detectionreagent can be adapted for use in western blot. Accordingly, thedetection reagent described herein can be adapted for use in variousapplications, e.g., but not limited to, pathogen detection and/oridentification, cancer-tissue analysis and other medical pathologyapplications, lineage tracking of differentiating stem cells, andlineage tracking and/or identification of dendritic cells.

Kits for various biological assays also provided herein. In someembodiments, a kit can comprise: (a) a plurality of the detectionreagents described herein or a portion thereof; and (b) at least onereagent. Examples of a reagent include, but are not limited to, areadout reagent, a wash buffer, a signal removal buffer, and anycombinations thereof.

The kits provided herein can be used for sequencing-based readout orhybridization-based readout. In some embodiments where the kit is usedfor hybridization-based readout, the kit can further comprise at leastone set of decoder probes complementary to at least a portion ofsubsequences of the detection reagents, wherein each subpopulation ofthe decoder probes comprises a different detectable label, eachdifferent detectable label producing a different signal signature.

In some embodiments, the kit can comprise a plurality of at least onecomponent of the detection reagents, e.g., the “nucleic acid label”component of the detection reagents. In such embodiments, users canattach the provided nucleic acid labels to their probe reagents ofinterest to form their own detection reagents described herein. In suchembodiments, the kit can further comprising at least one coupling agentthat allows the user to conjugate at least one nucleic acid label to theuser's probe reagents of interest. In other embodiments, the nucleicacid labels can be already attached to the pre-determined probe reagentsand are thus provided to users in the form of the detection reagentsthat are ready to use.

In some embodiments, the detection reagents provided in the kit orformed by a user can be provided in a solution phase. In otherembodiments, the detection reagents provided in the kit or formed by auser can be immobilized in a multi-well plate.

In some embodiments of any aspects described herein, the analytes ortarget molecules can be present in a solution phase. In someembodiments, the analytes or target molecules can be immobilized on asolid substrate or support.

To clarify, the compositions and methods described herein are differentfrom the ones described in the US Patent Application No.: US2007/0231824. The '824 application discusses methods of decoding asensor array containing immobilized microspheres, wherein themicrospheres are immobilized on a solid support (e.g., an arraysubstrate), rather than designed to be in a solution phase. As such, asample fluid is flowed over the sensor array containing immobilizedmicrospheres. The analytes in the sample fluid then bind to theimmobilized microspheres. After binding, the sample fluid is thendiscarded and the immobilized microspheres are analyzed. Accordingly,the compositions and the methods described in the '824 applicationcannot be used and detected directly on a sample (e.g., on a tissuesample) or in situ as described herein, e.g., immunofluorescence,immunohistochemistry, fluorescence in situ hybridization, or westernblot.

Further, the assay methods described herein are also different from thegeneral nanostring technology or other technologies as described in theU.S. Pat. No. 7,473,767, and the U.S. Patent Application No.: US2010/0047924. The general nanostring technology and nanoreporterdetection methods described in the '924 application are based ondetermination of the “spatial location of signals” emanating from thelabeled nanostrings or nanoreporters. The detection methods described inthe '767 patent is based on the color resulting from various ratios ofdifferent optical labels bound to the polynucleotide probes. All theseprevious methods will require at least a plurality of optical labels tobe detected simultaneously for determination of spatial location ofsignals or the resultant color from various ratios of different opticallabels. Accordingly, all these previous methods do not involve detectionof signals in a temporally-sequential manner as described herein.Further, there are at least two disadvantages of nanostring technologybased on detection of “spatial location of signals,” rather thantemporal detection of signals as described herein. First, compared tothe methods and detection reagents described herein, the nanostringtechnology generally requires very high optical magnification forspatially discerning separation of colors that are typically locatedvery close to each other within a nanostring; thus limiting a field ofview/sample size, and precision, and/or increasing instrument cost.Second, unlike the methods and detection reagents described herein, thenanostring technology generally requires a thorough control of theamount of probes used in detection, because too few probes would yield asignal that is difficult to be detected, but too many probes wouldincrease the likelihood of probes overlapping, and thus making thereadout impossible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows three different embodiments of the detection reagentsdescribed herein producing distinct sequencing readout.Pathogen-specific antibodies (e.g., anti-E-Coli, anti-S. aureus, andanti-C. albicans) are individually conjugated to a nanoparticle with atleast one nucleic acid label as described herein. In accordance with oneor more embodiments, the readout of the nucleic acid label can take theform of a set of optical images or spot-readings of, e.g., fluorescentor visible colors; the temporal sequence of optical images orspot-readings can then be computationally analyzed to determine theidentity of the corresponding probe reagent, e.g., a pathogen-specificantibody.

FIG. 2 shows three different embodiments of the detection reagentsdescribed herein producing distinct sequencing readout. DNAoligonucleotides complementary to target RNA expression (e.g., OCT4,SOX2, or KLF4) are individually conjugated to a nanoparticle with atleast one nucleic acid label as described herein. In accordance with oneor more embodiments, the readout of the nucleic acid label can take theform of a set of optical images or spot-readings of, e.g., fluorescentor visible colors; the temporal sequence of optical images orspot-readings can then be computationally analyzed to determine theidentity of the corresponding probe reagent, e.g., a DNA aptamerspecific for a RNA expression.

FIG. 3 shows exemplary forms of a nucleic acid label of the detectionreagent according to one or more embodiments described herein.

FIGS. 4A and 4B shows two exemplary configurations of probe reagents andnucleic acid labels on particles, in accordance with one or moreembodiments described herein.

FIG. 5 shows one embodiment of the detection reagents for an exemplaryhybridization-based readout method, in accordance with one or moreembodiments described herein. Pathogen-specific antibodies (e.g.,anti-C. albicans) are individually conjugated to a nanoparticlecomprising at least one nucleic acid label as described herein. Inaccordance with one or more embodiments, the readout of the nucleic acidlabel can be determined by hybridizing it with a small number of, e.g.,fluorescently-labeled decoder probes, imaging, and then advancing to thenext set of decoder probes. In order to allow the SeqTag to be read outquickly and without the use of enzymes or chemical reactions, the DNAoligonucleotide (SeqTag) is designed to include several hybridizationsites, each corresponding to a particular readout step. At each readoutstep, the sample is subjected to a mixture of fluorescently labeled DNAprobes that could potentially bind that step's hybridization site. Eachof the sites, however, is designed to bind only one of these probes,thus revealing the SeqTag's identifying code.

FIGS. 6A-6B show the readout images of superparamagnetic sphericalpolymer particles sold under the trade name DYNABEADS® beads (1 μm insize) localized on a sample using an exemplary hybridization-baseddetection method. Each bead was conjugated to one of 6 nucleic acidlabels, which in turn hybridized with a different set of decoder probesconjugated to either a green, red or blank optical label during eachreadout stage (FIG. 6A: Readout stage 1; FIG. 6B: Readout stage 2).

FIG. 7 is a set of images showing that each of the detection moleculeconstructs (SeqTag labels) properly stained the yeast and fluorescencedin accordance with three pre-determined sets of decoder probes. SeqTaglabels and oligonucleotide displacers were applied as follow:

-   -   Step 1: Set 1 Probes only;    -   Step 2: Set 1 Displacement oligonucleotides and Set 2 Probes;    -   Step 3: Set 2 Displacement oligonucleotides and Set 3 Probes;        and    -   Step 4: Set 3 Displacement oligonucleotides.        SeqTag Label and corresponding colors were as follow:    -   Set 1: SeqTag 1—Green, SeqTag 2—Red, and SeqTag 3—Blue;    -   Set 2: SeqTag 4—Green, SeqTag 5—Red, and SeqTag 6—Blue;    -   Set 3: SeqTag 7—Green and SeqTag 8—Red.

FIG. 8 shows SeqTag readout by a displacement-hybridization experimentaccording to an embodiment of the method described herein. SeqTag DNAlabels were incubated on a streptavidin-coated microarray. Thismicroarray was then exposed to a sequence of fluorescently labeleddetection probes and displacement oligonucleotidess, as perdisplacement-hybridization readout. Imaging the array after each readoutstep demonstrated fluorescence corresponding to the expected pattern(shown next to each image).

FIG. 9 is a schematic representation of displacement hybridizationaccording to an embodiment of the method. As the readout progresses,fluorescence from prior readout steps needst be removed so as not toobstruct the current step's signal. As illustrated in FIG. 9, this canbe accomplished using a displacement-hybridization method: each of theSeqTag's hybridization sites can be preceded by a short “toehold”sequence. During each readout step, the sample is subjected to a mixtureof “displacer” DNA oligonucleotides. One of these displacers iscomplementary to the preceding step's hybridization site and, with thehelp of the toehold, is capable of displacing the fluorescent probe thatwas bound there.

FIG. 10 is a schematic representation of a probe reagent according to anembodiment described herein. Shown is anoligonucleotide-antibody-streptavidin construct. A convenient andeffective way to SeqTag-label antibodies is through a streptavidinbridge. The antibody is biotinylated and the probe DNA oligonucleotides(SeqTag) are synthesized with a 5′-biotin modification. Then, by takingadvantage of streptavidin's native tetrameric form, three DNA strandsare bound to each antibody. As opposed to chemical conjugation methods,which can harm the antibody, this method proves gentle enough topreserve antibody function. As shown a single streptavidin molecule (2)is bound to three of the same DNA SeqTags (1) and a single antibody (3).

FIG. 11 is a schematic representation of detection of an analyte by theSeqTag labeled antibody shown in FIG. 10.

FIG. 12 is a schematic representation of detection reagents fordifferent analytes. As shown, different infectious agent (analytes) caneach be assigned their own SeqTag code to enable multiplexed detectionaccording to an embodiment described herein.

FIG. 13 is a schematic representation of detection reagents forSeqTagged Fluorescence in situ hybridization (FISH). FISH permitsmicrobes to be identified based on their ribosomal RNA sequence. In thecase of SeqTagged FISH, the FISH probe and its SeqTag can be synthesizedas a single DNA oligonucleotides as shown. Sequences shown are, from topto bottom, SEQ ID NO: 1 (5′-CCTACACACCAGCGTGCC-3′, probe for K.pneumonia); SEQ ID NO: 2 (5′-CCGCACTTTCATCTTCCG-3′, probe for H.influenza); and SEQ ID NO: 3 (GCCAAGGCTTATACTCGC, probe for C.albicans).

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods, detection reagents (or detection moleculesas used interchangeably herein) and kits for detecting a plurality ofanalytes in a sample. In accordance with embodiments of various aspectsdescribed herein, a probe reagent (e.g., antibody or aptamers) can bedirectly or indirectly labeled with a nucleic acid label. The nucleicacid information present on the nucleic acid label can then be decodedand/or detected in a temporally-sequential manner. The detectionreagents and methods described herein significantly increase the numberof different probes (and corresponding analytes) that can besimultaneously detected in a multiplex assay, as compared to antraditional assay where each probe is labeled with only fluorescentlabels or quantum dots, and thus multiplexing is limited by the numberof available and practically usable colors. Furthermore, because thedetection reagents described herein are detected and/or imaged in atemporal series of steps, the number of probes (and correspondinganalytes) that can be detected in a multiplex assay growsmultiplicatively with the number of detection steps in a time series andthe number of optical labels being used. By way of example only, 3 setof images in which 4 distinct optical labels are used can encode4×4×4=64 distinct probe reagents (e.g., antibodies).

Methods of Detecting a Plurality of Analytes in a Sample

One aspect of the inventions provides the methods for detecting aplurality of analytes in a sample, using the detection reagentsdescribed herein. The method includes (a) contacting the sample with acomposition comprising a plurality of detection reagents (which will bedescribed in detail later), wherein each subpopulation of the detectionreagents targets at least one different analyte; and (b) detecting in atemporally-sequential manner said plurality of the pre-determinedsubsequences of said detection reagents, wherein said detection of thesubsequences each generates a signal signature corresponding to saidsubsequence, and wherein a temporal order of the signal signaturescorresponding to said plurality of the subsequences of said detectionreagent identifies a subpopulation of the detection reagents. In someembodiments, the signal signature is a temporal signature. In someembodiments, the signal signature can further comprise a spatialsignature. A non-limiting example of a spatial signature includesspatial location of a signal signature.

In some embodiments, the temporal order of the signal signaturescorresponding to the plurality of the subsequences of the detectionreagent can be unique for each subpopulation of the detection reagents.In some embodiments, at least two or more signal signatures can be usedto identify the same subpopulation of the detection reagents.

In some embodiments, a detection reagent described herein can target atleast two (e.g., at least two, at least three or more) distinctanalytes. In some embodiments, a first subpopulation of the detectionreagents can target at least one analyte different from that of a secondsubpopulation of the detection reagents. By way of example only, a firstsubpopulation of the detection reagents can target at least analyte Aand analyte B, whereas a second subpopulation of the detection reagentscan target at least analyate B and analyte C. The readout of thesedetection reagents can be distinct but overlapping. Thus, differentanalytes can be identified by sampling them combinatorially anddetermining which one binds.

As used herein, the term “temporal order of the signal signatures”refers to a sequence of signal signatures determined in atemporally-sequential manner, i.e., the sequence of signal signatures isprogressed through by a number of active operations performed in atemporally-sequential manner, e.g., using a different set of decodingreagents or decoder probes in each active operation. In someembodiments, using a set of decoder probes in each active operation canyield one signal signature corresponding to one subsequence of thedetection reagents.

The composition comprising a plurality of detection reagents can existin any format. In some embodiments, the composition comprising aplurality of detection reagents can be in a form of a solution orsuspension comprising the detection reagents. In such embodiments, thecomposition can further comprise at least one agent. For example,without wishing to be bound, the agent can be a blocking buffer, asurfactant, unconjugated probe reagents, a stabilizer, an enzymeinhibitor, or any combinations thereof. In some embodiments where thecompositions are administered in vivo, the composition can furthercomprise a pharmaceutically-acceptable carrier. In other embodiments,the composition comprising a plurality of detection reagents can becontained or immobilized in a device (e.g., a syringe, or a microfluidicdevice) or an assay or reaction vessel (e.g., solid supports such asvials, and multi-well plates).

As used therein, the term “contacting” refers to any suitable means fordelivering, or exposing, a sample to a plurality of the detectionreagents described herein. In some embodiments, the term “contacting”refers to adding the detection reagents (e.g., suspended in a solution)directly to the sample. In some embodiments, the term “contacting” canfurther comprise mixing the sample with the detection reagents by anymeans known in the art (e.g., vortexing, pipetting, and/or agitating).In some embodiments, the term “contacting” can further compriseincubating the sample together with the detection reagents for asufficient amount of time, e.g., to allow binding of the probe reagentsto the target analytes. The contact time can be of any length, dependingon the binding affinities and/or concentrations of the probe reagentsand/or the analytes, concentrations of the detection reagents, and/orincubation condition (e.g., temperature). For example, the contact timecan be reduced if the sample and detection reagents are incubated at ahigher temperature. In some embodiments, the contact time between thesample and the detection reagents can be at least about 30 seconds, atleast about 1 minute, at least about 5 minutes, at least about 10minutes, at least about 15 minutes, at least about 30 minutes, at leastabout 1 hour, at least about 2 hours, at least about 3 hours, at leastabout 4 hours, at least about 6 hours, at least about 8 hours, at leastabout 10 hours, at least about 12 hours, at least about 24 hours, atleast about 48 hours or longer. One of skill in the art can adjust thecontact time accordingly.

For in vivo applications, the term “contacting” can refer toadministering the detection reagents to a subject, e.g., by oraladministration or by injection.

The sample can be contacted with at least one kind of the detectionreagents. In some embodiments, the sample can be contacted with at least2, at least 3, at least 4, at least 5, at least 6, at least 7, at least8, at least 9, at least 10, at least 15, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100 or more different kinds of the detection reagents. Insome embodiments, the sample can be contacted with at least 100, atleast 500, at least 1000, at least 5000, at least 10,000, at least50,000, at least 100,000 or more different kinds of the detectionreagents. Various kinds of the detection reagents described herein candiffer in types of probe reagents (e.g., nucleic acids vs. antibodies),target binding domains, and/or target analytes.

In some embodiments, the method described herein can further compriseprocessing the sample before contacting with the composition comprisinga plurality of detection reagents described herein. Depending on thetypes and/or natures of the samples and/or analytes, different sampleprocessing techniques can be used with the methods described herein.Exemplary sample processing techniques include, but are not limited to,mechanical processing of a sample (e.g., without limitations,homogenizing, centrifuging, vortexing, sectioning and shearing),addition of at least one reagent to a sample (e.g., without limitations,lysis buffers, RNA or DNA extraction reagents, RNA or DNA digestionreagents, enzyme inhibitors, fixing agents, organic solvents,antibodies, permeabilizing agents and immunohistochemistry agents),separation of a sample (e.g., without limitations, filtering,centrifuging, electrophoresis, western blot, and Northern blot),mounting a sample on a solid support (e.g., a microscopic slide), andany combinations thereof.

By way of example only, if a sample is a tissue from a subject (e.g., abiopsy for immunostaining), sample processing can include, but are notlimited to, tissue sectioning, mounting on a solid support, fixing thetissue, permeabilizing the tissue (if intracellular proteins are to bedetected), blocking non-specific reactions with the detection reagents.In some embodiments, proteins or nucleic acids can be isolated from atissue or fluid sample and then separated electrophoretically on aseparation medium (e.g., electrophoresis gel), followed by transferringthe proteins or nucleic acids to a blotting membrane. The blottingmembranes containing proteins or nucleic acids can then be contactedwith the detection reagents described herein. Methods of processingsamples before addition of various types of probe reagents for differentkinds of assays are well established in the art, and any of thosemethods can be performed prior to the contacting step of the methodsdescribed herein.

In some embodiments, the method described herein can further compriseremoving any unbound detection reagents before detection of thepre-determined subsequences in a temporally-sequential manner. The term“unbound detection reagents” as used herein refers to detection reagentsthat have not bound to or interacted with target analytes. The unbounddetection reagents can be removed from the sample by any methods knownin the art, e.g., rinsing with the sample with a buffered solution atleast once, at least two times, at least three times or above.

After the detection reagents bind to the target analytes in a sample,the nucleic acid labels of the detection reagents carrying nucleic-acidinformation can be decoded to allow identification of respective probereagent(s) conjugated to them, as opposed to traditional opticallabeling technologies, where an optical signature such as a fluorophoreis detected in the absence of providing any nucleic acid information. Inembodiments of various aspects described herein, the pre-determinedsubsequences within the nucleic acid labels are detected in atemporally-sequential manner. The term “temporally-sequential manner” isused in reference to detecting or decoding in a time series a pluralityof the pre-determined subsequences within the nucleic acid labels of anydetection reagents that are bound to target analytes in a sample. Insome embodiments, one or more pre-determined subsequences within atleast one nucleic acid label of each detection reagent can be detectedor decoded at each time point or detection step of a time series. Insome embodiments, one pre-determined subsequence within at least onenucleic acid label of each detection reagent can be detected or decodedat each time point or detection step of a time series. In someembodiments, at least one pre-determined subsequence (e.g., 1, 2, 3, 4,5, 6, or more pre-determined subsequences) at the same correspondinglocation within the nucleic acid label of each detection reagent can bedetected or decoded at each time point or detection step of a timeseries. The time period between any two time points or detection stepscan be of any length, e.g., seconds, minutes and hours. For example, thetime period between any two time points or detection steps can vary fromabout 5 seconds to about 2 hours, from about 10 seconds to about 1 hour,from about 30 seconds to about 30 mins, or from about 1 min to about 15mins. In some embodiments, the time period between any two time pointsor detection steps can be less than 5 seconds. In other embodiments, thetime period between any two time points or detection steps can be longerthan 2 hours, longer than 4 hours, longer than 6 hours, longer than 12hours, longer than 1 day. For example, a sample containing the detectionreagents can be maintained at room temperature, at a fridge temperature(e.g., between about 0° C. and about 10° C.) or at sub-zero temperatures(e.g., between −80° C. or lower and 0° C.) during the time periodbetween detection steps. In some embodiments, each subsequent detectionstep is performed substantially immediately one after another (e.g.,within less than 2 seconds, less than 1 second).

In some embodiments, the pre-determined subsequences can be detected inany temporal orders. In some embodiments, the next pre-determinedsubsequence to be detected after the previous one can be located closestto the previous one. In some embodiments, the next pre-determinedsubsequence to be detected after the previous one can be located atleast one, at least two, at least three, at least four, at least five ormore pre-determined subsequences apart from the previous one. In suchembodiments, any pre-determined subsequences that were bypassed in aprevious detection step can be detected afterward. In some embodimentsof the methods described herein, a computer-implemented software can beused to facilitate an analysis of the temporal readouts from thedetection steps, e.g., re-arranging the temporal readouts in an ordercorresponding to their spatial locations within the nucleic acid labelbefore further comparison and quantification analyses.

In some embodiments, the detection or decoding of the pre-determinedsubsequences can comprise nucleic acid sequencing. Methods forsequencing nucleic acids are well established to a skilled artisan,e.g., but not limited to ligation, hybridization, synthesis,amplification or single-base extension, or any combinations thereof. Byway of example only, as shown in FIG. 1 or FIG. 2, the nucleic acidlabels each contain three pre-determined subsequences (each of onenucleotide) conjugated together by a direct bond such as aphosphodiester bond. Each sequencing step decodes or determines onenucleotide, wherein each nucleobase (A, G, C or T) generates a distinctsignal signature corresponding to the nucleobase. Consequently, atemporal order or time series of the signal signatures generated fromeach sequencing step corresponds to the respective probe reagent, andthus identify the target analyte. Without wishing to be bound, in thisembodiment, the number of sequencing steps performed is not necessarilyequal to the number of the pre-determined sequences. In someembodiments, the number of sequencing steps performed can be less thanthe number of the pre-determined sequences. For example, as shown inFIG. 1, two sequencing steps could be sufficient to identify the threedifferent probe reagents, where each base is associated with a differentcolor. However, additional nucleic acid information can increase theaccuracy of identifying different probe reagents. Further, if thesequencing of each base can yield one of 4 colors, the n-basesubsequences (e.g., 3-base subsequences shown in FIG. 1 or FIG. 2) canproduce 4^(n) possible unique readouts, i.e., 4^(n) possible distinctprobe reagents can be distinguished using such detection reagentsdescribed herein.

While sequencing methods can convey single base difference, otherdetection or decoding methods that convey information by the presence orabsence of entire hybridization “sites” or pre-determined subsequenceson the nucleic acid label can also be used for the methods describedherein. In some embodiments, the detection step can comprise hybridizinga decoder probe with a subsequence on the nucleic acid label of thedetection reagent, wherein the decoder probe can comprise a detectablelabel. In particular embodiments, the detection method can comprise: (a)hybridizing a set of decoder probes with a subsequence of the detectionreagents, wherein each subpopulation of the decoder probes can comprisea detectable label, each detectable label producing a signal signature;(b) detecting said signal signature produced by the hybridization ofsaid set of decoder probes; and (d) repeating steps (a) and (b) forother subsequences of said detection reagents.

In some embodiments, each subpopulation of the decoder probes cancomprise a different detectable label, each different detectable labelproducing a different signal signature. In these embodiments, thedifferent signal signature produced by the hybridization of the set ofdecoder probes can be detected.

In some embodiments, each subpopulation of the decoder probes can becomplementary (e.g., partially complementary or completelycomplementary) to the subsequence of the detection reagents. In someembodiments, a first subpopulation and a second subpopulation of thedecoder probes can be complementary (e.g., partially complementary orcompletely complementary) to distinct subsequences of the detectionreagents. In some embodiments, at least two or more subpopulations ofthe decoder probes can bind to the same subsequence of the detectionreagents. For example, a first subpopulation and a second subpopulationof the decoder probes can be complementary (e.g., partiallycomplementary or completely complementary) to the same subsequence ofthe detection reagents.

By way of example only, FIG. 5 shows an exemplary detection reagentcomprising anti-C. albicans probe reagents and nucleic acid labelscontaining three hybridization sites or pre-determined subsequencesconjugated together by sequence linkers. In the first hybridizationstep, a first set of decoder probes each comprising a distinctdetectable label (e.g., complementary DNA readout probes shown in FIG.5, each comprising a distinct optical label) is hybridized with a firstpre-determined subsequence (e.g., Site 1 in FIG. 5), followed bydetection of a first signal signature produced by the hybridization. Inthe second hybridization step, a second set of decoder probes eachcomprising a distinct detectable label is hybridized with a secondpre-determined subsequence (e.g., Site 2 in FIG. 5), followed bydetection of a second signal signature produced by the hybridization.The second pre-determined subsequence can be the same or different fromthe first pre-determined subsequence. However, in preferred embodiments,the second pre-determined subsequence is different from the firstpre-determined subsequence, e.g., to minimize cross-hybridization witheach other. Accordingly, the hybridization and signal detection stepsare repeated for other subsequences of the detection reagents with adifferent set of decoder probes, thereby producing a temporal order ortime series of the signal signatures corresponding to the respectiveprobe reagent (and detection reagent).

As used herein, the term “decoder probe” refers an oligonucleotide witha sequence complementary to a pre-determined sequence of the nucleicacid label. By “complementary” is meant that a nucleic acid can formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. The decoderprobe sequence can be completely or partially complementary to apre-determined sequence. In some embodiments, partial complementarity isindicated by the percentage of contiguous residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “completelycomplementary” or 100% complementarity means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence. Lessthan perfect complementarity refers to the situation in which some, butnot all, nucleoside units of two strands can hydrogen bond with eachother.

The decoder probe can have a sequence of any length. In someembodiments, the decoder probe can have a sequence length of about 1 toabout 100 nucleotides, about 1 to about 50 nucleotides, about 2 to about50 nucleotides, about 5 to about 30 nucleotides, or about 5 to about 20nucleotides.

In some embodiments, the decoder probe can comprise at least onedetectable label described herein. In some embodiments, the detectablelabel can be an optical label selected from the group consisting of asmall-molecule dye, a fluorescent molecule or protein, a quantum dot, acolorimetric reagent, a chromogenic molecule or protein, a Raman label,and any combinations thereof. In some embodiments, the detectable labelor optical label can be a fluorescent molecule or protein.

In some embodiments, the decoder probe can be modified, e.g., basemodification or activated with a functional group for linkage to adetectable label.

The number of decoder probes in each set can vary, depending on thenumber of distinct subsequences in each hybridization. In someembodiments, there can be about 1 to about 100 decoder probes, about 2to about 50 decoder probes, about 4 to about 20 decoder probes in eachset. In some embodiments, there can be about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more decoderprobes in each set. In some embodiments, while each subsequence site canhybridize with a large number of decoder probes, each set of decoderprobes added in each readout step to hybridize with the subsequence sitewithin the detection reagents can generally have as many as the numberof available fluorescent colors. For example, each set of the decoderprobes added in each readout step to hybridize with the subsequence siteof the detection reagents can contain about 3-4 decoder probes, each ofwhich is labeled with a distinct fluorescent color. In the case of usingquantum dots or Raman labels as detection labels, there can be more than3-4 decoder probes in each set added during each readout step.

Without wishing to be bound, an example of “non-overlapping nucleic acidlabels” or “non-overlapping SeqTag labels” (i.e., no two decoder probeswill hybridize with the same spatial site of the nucleic acid label) isshown herein for illustrative purposes. Assuming there are 12pre-determined subsequences (e.g., nucleic acid sequences) designed forminimal cross-hybridization with each other and each others' complements(e.g., A1, A2, A3, A4, B1, B2, B3, B4, C1, C2, C3, and C4). Consider adetection reagent comprising a nucleic acid label of the form:

-   -   5′-A[1-4]-B[1-4]-C[1-4]-3′

where each position (e.g., A[1-4]) holds only one of the subsequences(e.g., A2). Now, this subsequence is decoded or detected by usingdecoder probes or complementary probes A1*-A4* each labeled in one offour fluorescent colors. After readout, the signal produced by thefluorophore can be removed (e.g., denaturing to undo the hybridization(and thus removing the fluorophore)) before continuing with B1*-B4*.Since each step yields one of four outcomes, this coding scheme canprovide 4×4×4=64 unique readouts. These hybridization-based probes canbe used to label 64 different probe reagents that can be read out inthree cycles.

In the case where two decoder probes can overlap, a single site (e.g., Ain the above nucleic acid label form), which can accept one of thedifferent decoder probes, can be used. This single site can be read outby subjecting it to the different decoder probes, e.g., in sets of 4using the nucleic acid label form as shown above. When the correct setis reached, the nucleic acid label, e.g., SeqTag label, should be dark(no-color). This example is not construed to be limiting and anymodifications apparent to one of skill in the art is also within thescope of the inventions.

The advantage of readout by hybridization is that it can be quick:hybridization can take place in minutes or less. Furthermore, nochemistry or enzymes are required during the readout process, as thereadout process can be performed by similar methods as used in nucleicacid sequencing, microscopy, spectroscopy, or any combinations thereof.Thus, hybridization-based readout method can reduce cost, reduce reagentstorage and/or simplify the process.

In some embodiments of the methods described herein, there can be nolimit in the spatial movement of an analyte in a sample during atemporal detection of the detection reagents, for example, provided thatthe analyte stay within the field of detection and there is at least onesame distinguishable feature in each image taken during a temporaldetection so that the images can be aligned to each other based on thesame distinguishable feature. In some embodiments where there is no suchdistinguishable feature, the spatial movement of an analyte in a samplecan be less than 100 μm, including less than 50 μm, less than 25 μm,less than 10 μm, less than 1 μm or smaller, over a time period, duringwhich a temporal detection of the detection reagents occurs. In someembodiments, the spatial movement of an analyte in a sample can be lessthan 1000 nm, including less than 500 nm, less than 250 nm, less than100 nm, less than 50 nm, less than 10 nm or smaller, over a time period,during which a temporal detection of the detection reagents occurs. Moreimportantly, the spatial movement limit of an analyte in a sample duringa temporal detection is determined by the ability of matchingdistinguishable features between images taken during a temporaldetection, which can be affected by imaging conditions. In someembodiments, the analyte can be fixed on a solid substrate or support.In some embodiments where there is or expects to be a spatial movementof an analyte during temporal detection, the location of the analytewith respect to a sample during each detection step can be determinedand registered. Such spatial shift can then be corrected afterwardduring signal analysis using any art-recognized computer-implementedalgorithms.

The length of time required to perform a temporal detection of thedetection reagents (e.g., the length of time it takes to obtain atemporal sequence of signal signatures for the detection reagents) canvary, depending on the number of pre-determined subsequences to bedetected or read and/or the number of available detection signals (e.g.,fluorescent color and/or brightfield) to be read. In some embodiments,the length of time required to perform a temporal detection of thedetection reagents can be, for example, but not limited to, 5 seconds,10 seconds, 15 seconds, 20 seconds, 30 seconds, 1 mins, 2 mins, 3 mins,4 mins, 5 mins, 15 mins, 30 mins, 1 hour, 2 hours, 4 hours, 6 hours, orlonger.

The detection reagents can be detected by any means available in the artthat is capable of detecting the specific signals on a given detectionreagent generated during sequencing- or hybridization-based methods.Where the detection reagents (e.g., hybridized with decoder probes) arefluorescently labeled, suitable consideration of appropriate excitationsources can be readily determined. Possible sources can include but arenot limited to arc lamp, xenon lamp, lasers, light emitting diodes orsome combination thereof. The appropriate excitation source is used inconjunction with an appropriate optical detection system, for example aninverted fluorescent microscope, an epi-fluorescent microscope or aconfocal microscope. Preferably, a microscope is used that can allow fordetection with enough spatial resolution to separate distinct signalsfrom individual detection reagents.

Exemplary methods for detection of the detection reagents that areapplicable to the methods described herein include, without limitations,the methods described in U.S. Pat. No. 7,473,767, US patent publicationno. 2007/0166708, and US application number US 2010/0261026, all ofwhich are incorporated by reference herein in its entirety.

Additional methods that can be used to detect optical signaturesinclude, but are not limited to, any spectroscopic techniques, flowcytometry, or any art-recognized methods involving an optical scannerand/or a photodetector (e.g., without limitations, a charge-coupleddevices, active pixel sensors, photodiode light sensors (e.g., LEDs),optical detectors, and any combinations thereof). Non-limiting examplesof spectroscopic techniques can include absorption spectroscopy,emission spectroscopy, elastic scattering spectroscopy, reflectionspectroscopy, impedance spectroscopy, inelastic spectroscopy, coherentor resonance spectroscopy, surface plasmon fluorescence spectroscopy,Raman spectroscopy, and any combinations thereof. Spectroscopytechniques can be used to detect light of any wavelengths, including,but not limited to, microwave, terahertz, infrared, near infrared,visible, ultraviolet, x-ray, gamma, and any combinations thereof.

Without wishing to be limited, in some embodiments, at least onepre-determined subsequence (e.g., individual bases or hybridizationregions) can correspond to no optical signature; that is the absence ofcolor can be considered as an additional color. In other embodiments, atleast one pre-determined subsequence (e.g., individual bases orhybridization regions) can correspond to a compound optical signature,e.g., two or more simultaneous fluorescence in multiple channels duringdetection (e.g., by microscopy).

While a single area of a sample can interact with more than one probereagents, the nucleic acid label can be designed such that any known orpotential overlaps could be teased apart from the signal output. In thecase where any probe may potentially overlap with all others, one canuse the readout-by-hybridization variation and assign each probe asingle unique hybridization sequence. Such approach can avoid multiplelengthy probe incubations and damaging stripping steps.

In some embodiments of the methods described herein, the signalsignatures produced during any readout step (e.g., sequencing-based orhybridization-based) should be removed before advancing to the nextpre-determined subsequence of the detection reagents. The removal of thesignal signatures can be done by any methods known in the art,including, but not limited to, washing, heating, photo-bleaching,displacement, cleavage, enzymatic digestion, quenching, chemicaldegradation, bleaching, oxidation, and any combinations thereof.

In some embodiments, the decoder probes can be designed such that theycan be simply washed out either with a plain buffer, or they can bemodified by varying salt concentrations or using detergents ordenaturants such as formamide or dimethyl sulfoxide (DMSO).

In other embodiments, the fluorescence or color signature of a readoutstep can be attenuated or eliminated by photo-bleaching the signal usingsufficient optical exposure. In alternative embodiments, thefluorescence or color signature of a readout step can be attenuated oreliminated by subjecting the fluorescence or color signature to chemicaldegradation under appropriate conditions, e.g., using a reducing agentor oxidizing solution such as 0.01 M sodium periodate.

In some embodiments, the decoder probes can be displaced from theirhybridization sites by introducing other reagents or probe displacersthat have stronger binding affinities to those same sites. This can bedone, for example, by using nucleic acid sequences that are longer thanand/or have better complementarity than the decoder probe sequences. Forexample, to create “better complementarity” of the probe displacers, insome embodiments, mismatches can be seeded in the hybridization region.In other embodiments, the hybridization region can be preceded and/orpost-ceded with a “toe-hold” of around 3-8 bases (e.g., 6 bases). Insuch embodiments, the “better complementarity” of the probe displacercan be outside of the hybridization region, which can make the design ofthe hybridization regions easier.

In some embodiments, enzymes can be used to displace, digest, cut and/orcleave the detectable labels, the decoder probe sequence, the hybridizedcomplex (formed by the decoder probe and pre-determined subsequence),and/or the cleavable sequence linker to the hybridized complex, in orderto remove the signal signatures. One example is to introduce adeoxyuridine into the decoder probe. This modified base can be cleavedusing the enzyme mix known as USER, thereby cutting the decoder probesequence into two parts. Since each part is now shorter, it ischaracterized by a lower melting temperature and can melt off thehybridization sites of the detection reagents. Alternatively, one ofskill in the art can employ one of numerous art-recognizedsequence-specific nucleases or restriction enzymes, which can cut eitherthe decoder probe sequence, the pre-determined subsequence that has beenhybridized with decoder probes, the cleavable sequence linker attachedto the hybridized subsequence and/or the hybridized complex thereof,thereby removing the signal signature.

In some embodiments, thermal denaturing can be used to remove the signalsignature from a previous readout. In some embodiments where sequencingis involved, thermal denaturing can be reduced or avoided by using asequencing-by-ligation approach and by setting the nucleic acid labelbase that immediately follows the sequencing primer (in the direction ofligation) to an adenine. Correspondingly, the ligation probes shouldthen include a (deoxy-)uracil in the primer-proximal position. Withoutwishing to be bound by theory, an enzyme such as USER, which cleaves DNAat uracils can be used to remove the ligation probe and ready the systemfor the next sequencing step. These fragments will have lower meltingtemperatures than their parent probes, and these temperatures can bedesigned to fall below the operating temperature (or require anacceptable denaturing temperature).

After detection of the pre-determined subsequences is completed in atemporally-sequential manner, in some embodiments, the method describedherein can further comprise comparing the temporal order of the signalsignatures with different identifiers of said at least one probereagent, wherein an agreement between the temporal order of the signalsignatures and a particular identifier of said at least one probereagent identifies the analyte in the sample. In some embodiments, themethod can further comprise measuring the intensity of the signalsignatures generated from each subpopulation of the detection reagents.In some embodiments, the intensity of the signal signatures generatedfrom each subpopulation of the detection reagents can indicate an amountof the analyte. In some embodiments, the relative intensity of thesignal signatures can be used in identification of each subpopulation ofthe detection reagents. Thus, the intensity of the signal signatures canbe used as part of a coding scheme of the detection reagents describedherein. The comparing and intensity measuring steps can be performed,e.g., by a computer-implemented software or algorithm.

Types of signal signature(s) can vary upon different embodiments ofdetection reagents and/or decoder probes described herein. As usedherein, the term “signal signature” refers to a change in, or occurrenceof, a response or indicator that is detectable either by observation orinstrumentally. In certain instances, the signal signature isfluorescence or a change in fluorescence, e.g., a change in fluorescenceintensity, fluorescence excitation or emission wavelength distribution,fluorescence lifetime, and/or fluorescence polarization. By way ofexample only, the fluorescence can be produced by binding a fluorophoreto a decoder probe, and/or by detecting the hybridization using afluorescent dye, e.g., a proprietary unsymmetrical cyanine dye soldunder the trade name SYBR® Gold, that lights up when nucleic acidsequence becomes double-stranded. In certain other instances, the signalsignature can be radioactivity (i.e., radiation), including alphaparticles, beta particles, nucleons, electrons, positrons, neutrinos,and gamma rays emitted by a radioactive substance such as aradionuclide. By way of example, the detection reagents and/or decoderprobes can comprise an optical molecule or label, thus producing opticalsignatures. Examples of optical signatures can include, withoutlimitations, signatures of fluorescent color, visible light, no-color,and any combinations thereof. In such embodiments, the opticalsignatures can be detected by optical imaging or spectroscopy.

Detection Reagents (or Detection Molecules as Used InterchangeablyHerein)

Another aspect provided herein is a detection reagent, which can be, forexample, used in the methods described herein for any multiplexingassays. The detection reagent comprises at least one probe reagent andat least one nucleic acid label, wherein said at least one nucleic acidlabel comprises at least one pre-determined subsequence to be detectedin a temporally-sequential manner; wherein said at least onepre-determined subsequence forms an identifier of said at least oneprobe reagent; and wherein said at least one probe reagent and said atleast one nucleic acid label are conjugated together.

The detection reagents described herein can exist in different forms. Byway of example only, in some embodiments, the detection reagent can be adetection molecule. In some embodiments, the detection reagent can be adetection particle. In some embodiments, the detection reagent can bemulti-molecular.

As used herein, the term “conjugated” refers to two molecules beinglinked to each other, e.g., attaching a probe reagent to a nucleic acidlabel. The conjugation process can be performed, e.g., via a chemicalreaction, or via a linker, which will be described later.

Depending on various applications and/or assay conditions (e.g.,sensitivity, sample volume/concentration), a readout signal of adetection reagent can be amplified by increasing the number of thenucleic acid labels present in the detection reagent, e.g., byconjugating at least one probe reagent to a plurality of nucleic acidlabels. In such embodiments, a plurality of the nucleic acid labelspresent in the detection reagent can range from about 2 to about100,000, about 2 to about 10,000, about 2 to about 1,000, or about 2 toabout 100. In some embodiments where the detection reagent comprises aparticle as a hub, the number of possible nucleic acid labels present inthe detection reagent can depend on the size of a particle. Generally,the larger the particle it is, the more nucleic acid labels can beincorporated into the detection reagent. For example, a particle ofabout 1-2 μm in size can allow incorporation of about 100,000 nucleicacid labels into the detection reagent. In some embodiments, there canbe 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,50, 100, 500, 1000, 5000, 10000, 50000, 100000 nucleic acid labelspresent in the detection reagent. One of skill in the art can determinethe optimum number of nucleic acid labels present the detection reagentwithout any undue experimentation.

The detection reagents described herein can be used in any biologicalassays for detection, identification and/or quantification of targetmolecules or analytes, including counting marked cells such as bacteriaor cancer cells, in a sample. By way of example only, in someembodiments, the detection reagent can be adapted for use inimmunofluorescence. In alternative embodiments, the detection reagentcan be adapted for use in immunohistochemistry. In other embodiments,the detection reagent can be adapted for use in fluorescence in situhybridization. In some embodiments, the detection reagent can be adaptedfor use in western blot. Depending on the nature of the sample and/orapplications, the detection reagent can be adapted to be in any format,e.g., immobilized on a solid support, or in a solution or suspensionphase. In certain embodiments, the detection reagent can be adapted tobe present in a solution or suspension phase. The phrase “in a solutionor suspension phase” as used herein generally refers to suspending thedetection reagents in a liquid fluid, e.g., an aqueous buffer solution.Additional applications of the detection reagents and/or methodsdescribed herein will be discussed.

Probe Reagents (or Probe Molecules as Used Interchangeably Herein)

Each of the detection reagents described herein can comprise any numberof probe reagents. In some embodiments, the detection reagent cancomprise one or more probe reagents, e.g., at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10 or more probe reagents. In one embodiment, thedetection reagent can comprise one probe reagent. In other embodiments,the detection reagent can comprise a plurality of probe reagents, e.g.,ranging from about 2 to about 100,000 probe reagents, about 2 to about10,000 probe reagents, about 2 to about 1,000 probe reagents, or about 2to about 100 probe reagents. In some embodiments where the detectionreagent comprises a particle as a hub, the number of possible probereagents present in the detection reagent can depend on the size of aparticle. Generally, the larger the particle it is, the more probereagents can be incorporated into the detection reagent. For example, aparticle of about 1-2 μm in size can allow incorporation of about100,000 probe reagents into the detection reagent. In some embodiments,there can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 50, 100, 500, 1000, 5000, 10000, 50000, 100000 probereagents present in the detection reagent. One of skill in the art candetermine the optimum number of probe reagents present the detectionreagent without any undue experimentation.

As used interchangeably herein, the term “probe,” “probe reagent” or“probe molecule” refers to an entity (e.g., but not limited to, amolecule, a particle, a composite entity, or a multi-molecular entity)that interacts with or binds to a target molecule or an analyte for theanalysis of the target or the analyte. Typically the nature of theinteraction or binding is noncovalent, e.g., by hydrogen, electrostatic,or van der Waals interactions, however, binding can also be covalent.Probe reagents can be entities (e.g., but not limited to, molecules, aparticles, composite entities, or multi-molecular entities) capable ofundergoing binding or molecular recognition events with targetmolecules. Probe reagents can be naturally-occurring, recombinant orsynthetic. Examples of the probe reagent can include, but are notlimited to, a nucleic acid, an antibody or a portion thereof, anantibody-like molecule, an enzyme, a cell, an antigen, a small molecule,a protein, a peptide, a peptidomimetic, an aptamer, and any combinationsthereof. By way of example only, in immunohistochemistry, the probereagent can include an antibody specific to the target antigen to beanalyzed. An ordinary artisan can readily identify appropriate probereagents for the target molecules or analytes of interest to be detectedin various bioassays. In some embodiments, the probe reagent can bemulti-molecular. For example, in one embodiment, the probe reagent cancomprise a particle, an antibody, biotin and/or streptavidin, or anycombinations thereof.

In some embodiments, the probe reagents can be modified by any meansknown to one of ordinary skill in the art. Methods to modify each typeof probe reagents are well recognized in the art. Depending on the typesof probe reagents, an exemplary modification includes, but is notlimited to genetic modification, biotinylation, labeling (for detectionpurposes), chemical modification (e.g., to produce derivatives orfragments of the probe reagent), and any combinations thereof. In someembodiments, the probe reagent can be genetically modified. In someembodiments, the probe reagent can be biotinylated.

As used herein, the terms “proteins” and “peptides” are usedinterchangeably herein to designate a series of amino acid residuesconnected to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and “peptide”,which are used interchangeably herein, refer to a polymer of proteinamino acids, including modified amino acids (e.g., phosphorylated,glycated, etc.) and amino acid analogs, regardless of its size orfunction. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “peptide” as used herein refers to peptides, polypeptides, proteinsand fragments of proteins, unless otherwise noted. The terms “protein”and “peptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary peptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

As used herein, the term “peptidomimetic” refers to a molecule capableof folding into a defined three-dimensional structure similar to anatural peptide

The term “nucleic acids” used herein refers to polymers(polynucleotides) or oligomers (oligonucleotides) of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar linkages. The term “nucleic acid” also includes polymers oroligomers comprising non-naturally occurring monomers, or portionsthereof, which function similarly. Exemplary nucleic acids include, butare not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA),locked nucleic acid (LNA), peptide nucleic acids (PNA), and polymersthereof in either single- or double-stranded form. Locked nucleic acid(LNA), often referred to as inaccessible RNA, is a modified RNAnucleotide. The ribose moiety of an LNA nucleotide is modified with anextra bridge connecting the 2′ oxygen and 4′ carbon. The bridge “locks”the ribose in the 3′-endo conformation. LNA nucleotides can be mixedwith DNA or RNA residues in the oligonucleotide whenever desired. SuchLNA oligomers are generally synthesized chemically. Peptide nucleic acid(PNA) is an artificially synthesized polymer similar to DNA or RNA. DNAand RNA have a deoxyribose and ribose sugar backbone, respectively,whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycineunits linked by peptide bonds. PNA is generally synthesized chemically.Unless specifically limited, the term “nucleic acids” encompassesnucleic acids containing known analogs of natural nucleotides, whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer, et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608(1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)). Theterm “nucleic acid” should also be understood to include, asequivalents, derivatives, variants and analogs of either RNA or DNA madefrom nucleotide analogs, and, single (sense or antisense) anddouble-stranded polynucleotides.

In some embodiments, the term “nucleic acid” described herein caninclude a modified nucleic acid. Modified nucleic acids are well knownin the art. Thus, a nucleic acid described herein can comprise one ormore nucleic acid modifications known in the art. For example, thenucleic acid can comprise one or more nucleic acid modificationsselected from the group consisting of internucleotide linkagemodifications (intersugar linkage modifications), sugar modifications,nucleobase modifications, backbone modifications/replacements, and anycombinations thereof. Exemplary internucleotide linkage modificationsinclude, but are not limited to, phosphorothioate, phosphorodithioate,phosphotriester (e.g. alkyl phosphotriester), aminoalkylphosphotriester,alkyl-phosphonate (e.g., methyl-phosphonate), selenophosphate,phosphoramidate (e.g., N-alkylphosphoramidate), boranophosphonate, andthe like. Exemplary sugar modifications include, but are not limited to,2′-O-Me (2′-O-methyl), 2′-O-MOE (2′-O-methoxyethyl), 2′-F,2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), 2′-S-methyl,2′-O—CH₂-(4′-C) (LNA), 2′-O—CH₂CH₂-(4′-C) (ENA), 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), arabinose sugar, and the like. Exemplary nucleobasemodifications include, but are not limited to, inosine, xanthine,hypoxanthine, nubularine, isoguanisine, tubercidine, 5-methylcytosine(5-me-C); 5-hydroxymethyl cytosine; xanthine; hypoxanthine;2-aminoadenine; 6-methyl and other 6-alkyl derivatives of adenine andguanine; 2-propyl and other 2-alkyl derivatives of adenine and guanine;2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-propynyl uracil;5-propynyl cytosine; 6-azouracil; 6-azocytosine; 6-azothymine; 5-uracil(pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines; 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines; 7-methyl and other 7-alkyl derivatives of adenine andguanine; 8-azaguanine; 8-azaadenine; 7-deazaguanine; 7-deazaadenine;3-deazaguanine; 3-deazaadenin; universal base; and any combinationsthereof. Exemplary backbone modifications include, but are not limitedto, morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA),aminoethylglycyl PNA (aegPNA), backnone-extended pyrrolidine PNA(bepPNA), and the like.

The term “enzymes” as used here refers to a protein molecule thatcatalyzes chemical reactions of other substances without it beingdestroyed or substantially altered upon completion of the reactions. Theterm can include naturally occurring enzymes and bioengineered enzymesor mixtures thereof. Examples of enzyme families include kinases,dehydrogenases, oxidoreductases, GTPases, carboxyl transferases, acyltransferases, decarboxylases, transaminases, racemases, methyltransferases, formyl transferases, and α-ketodecarboxylases.

As used herein, the term “aptamers” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules. In some embodiments,the aptamer recognizes the non-oligonucleotide molecule or group ofmolecules by a mechanism other than Watson-Crick base pairing or triplexformation. Aptamers can include, without limitation, defined sequencesegments and sequences comprising nucleotides, ribonucleotides,deoxyribonucleotides, nucleotide analogs, modified nucleotides andnucleotides comprising backbone modifications, branchpoints andnonnucleotide residues, groups or bridges. Methods for selectingaptamers for binding to a molecule are widely known in the art andeasily accessible to one of ordinary skill in the art.

As used herein, the term “antibody” or “antibodies” refers to an intactimmunoglobulin or to a monoclonal or polyclonal antigen-binding fragmentwith the Fc (crystallizable fragment) region or FcRn binding fragment ofthe Fc region. The term “antibodies” also includes “antibody-likemolecules”, such as fragments of the antibodies, e.g., antigen-bindingfragments. Antigen-binding fragments can be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intact antibodies.“Antigen-binding fragments” include, inter alia, Fab, Fab′, F(ab′)₂, Fv,dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single domain antibodies, chimericantibodies, diabodies, and polypeptides that contain at least a portionof an immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide. Linear antibodies are also included for thepurposes described herein. The terms Fab, Fc, pFc′, F(ab′) 2 and Fv areemployed with standard immunological meanings (Klein, Immunology (JohnWiley, New York, N.Y., 1982); Clark, W. R. (1986) The ExperimentalFoundations of Modern Immunology (Wiley & Sons, Inc., New York); andRoitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell ScientificPublications, Oxford)). Antibodies or antigen-binding fragments specificfor various antigens are available commercially from vendors such as R&DSystems, BD Biosciences, e-Biosciences and Miltenyi, or can be raisedagainst these cell-surface markers by methods known to those skilled inthe art.

As used herein, the term “Complementarity Determining Regions” (CDRs;i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop.

The expression “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The expression “single-chain Fv” or “scFv” antibody fragments, as usedherein, is intended to mean antibody fragments that comprise the VH andVL domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. (Plückthun, ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994)).

The term “diabodies,” as used herein, refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy-chainvariable domain (VH) Connected to a light-chain variable domain (VL) inthe same polypeptide chain (VH-VL). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. (EP 404,097; WO 93/11161; Hollinger etah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).

As used herein, the term “small molecules” refers to natural orsynthetic molecules including, but not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,organic or inorganic compounds (i.e., including heteroorganic andorganometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

The term “cells” used herein refers to any cell, prokaryotic oreukaryotic, including plant, yeast, worm, insect and mammalian.Mammalian cells include, without limitation; primate, human and a cellfrom any animal of interest, including without limitation; mouse,hamster, rabbit, dog, cat, domestic animals, such as equine, bovine,murine, ovine, canine, feline, etc. The cells may be a wide variety oftissue types without limitation such as; hematopoietic, neural,mesenchymal, cutaneous, mucosal, stromal, muscle spleen,reticuloendothelial, epithelial, endothelial, hepatic, kidney,gastrointestinal, pulmonary, T-cells etc. Stem cells, embryonic stem(ES) cells, ES-derived cells and stem cell progenitors are alsoincluded, including without limitation, hematopoeitic, neural, stromal,muscle, cardiovascular, hepatic, pulmonary, gastrointestinal stem cells,etc. Yeast cells may also be used as cells in some embodiments of themethods described herein. In some embodiments, the cells can be ex vivoor cultured cells, e.g. in vitro. For example, for ex vivo cells, cellscan be obtained from a subject, where the subject is healthy and/oraffected with a disease. Cells can be obtained, as a non-limitingexample, by biopsy or other surgical means know to those skilled in theart.

As used herein, the term “antigens” refers to a molecule or a portion ofa molecule capable of being bound by a selective binding agent, such asan antibody, and additionally capable of being used in an animal toelicit the production of antibodies capable of binding to an epitope ofthat antigen. An antigen may have one or more epitopes. The term“antigen” can also refer to a molecule capable of being bound by anantibody or a T cell receptor (TCR) if presented by MHC molecules. Theterm “antigen”, as used herein, also encompasses T-cell epitopes. Anantigen is additionally capable of being recognized by the immune systemand/or being capable of inducing a humoral immune response and/orcellular immune response leading to the activation of B- and/orT-lymphocytes. This may, however, require that, at least in certaincases, the antigen contains or is linked to a Th cell epitope and isgiven in adjuvant. An antigen can have one or more epitopes (B- andT-epitopes). The specific reaction referred to above is meant toindicate that the antigen will preferably react, typically in a highlyselective manner, with its corresponding antibody or TCR and not withthe multitude of other antibodies or TCRs which may be evoked by otherantigens. Antigens as used herein may also be mixtures of severalindividual antigens.

In some embodiments, the probe reagent can be an antibody or a portionthereof, or an antibody-like molecule. In such embodiments, the probereagents can be used to, for example, detect and/or identify pathogentype or speices, the presence of cell or disease markers, cellularprotein expression levels, phosphorylation or other post-translationmodification state, or any combinations thereof. By way of example only,FIG. 1 shows three different embodiments of the detection reagentscomprising at least one (e.g., 1, 2, 3, 4, 5 or more) pathogen-specificantibodies (e.g., anti-E. Coli, anti-S. aureus, and anti-C. albicans).

In some embodiments, the probe reagent can be a nucleic acid (e.g., DNA,RNA, LNA, PNA, or any combinations thereof). In such embodiments, thenucleic acids can be used to determine, for example, the existence ofcharacteristic cellular DNA or RNA sequences (such as in fluorescent insitu hybridization), RNA expression levels, miRNA presence andexpression, and any combinations thereof, in various applications, e.g.,for pathogen detection and/or identification.

In some embodiments, the probe reagent can be a protein or a peptide. Insuch embodiments, the protein or peptide can be essentially any proteinswith known binding targets. Examples include, but are not limited to,innate-immune proteins (e.g., without limitations, MBL, Dectin-1, TLR2,and TLR4 and any proteins disclosed in U.S. Provisional Application No.:61/508,957, the content of which is incorporated herein by reference inits entirety) and proteins comprising the chitin-binding domain. Suchinnate-immune proteins and chitin-binding domain proteins can be used todetect their corresponding pattern-recognition targets (e.g., microbessuch as bacteria) and fungus, respectively. By way of example only,instead of using pathogen-specific antibodies as probe reagents in thedetection reagents as shown in FIG. 1, innate-immune proteins (e.g.,MBL) or chitin-binding domain proteins can be used as probe reagents fordetection of pathogens. While such detection reagents can be used todetect pathogens, they may not be pathogen-specific, as compared to theones using pathogen-specific antibodies as probes molecules.

In some embodiments, the probe reagent can be an aptamer. In someembodiments, the probe reagent can be a DNA or RNA aptamer. The aptamerscan be used in various bioassays, e.g., in the same way as antibodies ornucleic acids described herein. By way of example only, FIG. 2 showssome exemplary embodiments of the detection reagents comprising at leastone (e.g., 1, 2, 3, 4, 5 or more) DNA aptamers (e.g., with a nucleotidesequence complementary to nuclear reprogramming factors, such as Oct4,Sox2, and Klf4). Such detection reagents can be used to determine RNAexpression level of nuclear reprogramming factors in somatic cells fordetecting, screening, or identifying stem cells (e.g., inducedpluripotency stem cells).

In some embodiments, the probe reagent can be a cell surface receptorligand. As used herein, a “cell surface receptor ligand” refers to amolecule that can bind to the outer surface of a cell. Exemplary, cellsurface receptor ligand includes, for example, a cell surface receptorbinding peptide, a cell surface receptor binding glycopeptide, a cellsurface receptor binding protein, a cell surface receptor bindingglycoprotein, a cell surface receptor binding organic compound, and acell surface receptor binding drug. Additional cell surface receptorligands include, but are not limited to, cytokines, growth factors,hormones, antibodies, and angiogenic factors.

When the detection reagents described herein are used as targeteddelivery vehicles, e.g., for a diagnostic agent, in some embodiments,the probe reagent can be an endosomolytic ligand. As used herein, theterm “endosomolytic ligand” refers to molecules having endosomolyticproperties. Endosomolytic ligands can promote the lysis of and/ortransport of the composition described herein, or its components, fromthe cellular compartments such as the endosome, lysosome, endoplasmicreticulum (ER), Golgi apparatus, microtubule, peroxisome, or othervesicular bodies within the cell, to the cytoplasm of the cell. Someexemplary endosomolytic ligands include, but are not limited to,imidazoles, poly or oligoimidazoles, linear or branchedpolyethyleneimines (PEIs), linear and branched polyamines, e.g.spermine, cationic linear and branched polyamines, polycarboxylates,polycations, masked oligo or poly cations or anions, acetals,polyacetals, ketals/polyketals, orthoesters, linear or branched polymerswith masked or unmasked cationic or anionic charges, dendrimers withmasked or unmasked cationic or anionic charges, polyanionic peptides,polyanionic peptidomimetics, pH-sensitive peptides, natural andsynthetic fusogenic lipids, natural and synthetic cationic lipids.

In other embodiments, the probe reagent for use in delivery of an agent(e.g., a diagnostic agent) encapsulated within the detection reagentsdescribed herein can be a PK modulating ligand. As used herein, theterms “PK modulating ligand” and “PK modulator” refers to moleculeswhich can modulate the pharmacokinetics of the composition describedherein. Some exemplary PK modulator include, but are not limited to,lipophilic molecules, bile acids, sterols, phospholipid analogues,peptides, protein binding agents, vitamins, fatty acids, phenoxazine,aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen,carprofen, PEGs, biotin, and transthyretia-binding ligands (e.g.,tetraiidothyroacetic acid, 2,4,6-triiodophenol and flufenamic acid).

In various embodiments, the detection reagent described herein cancomprise one kind/species of probe reagents or different kinds/speciesof probe reagents. In some embodiments, the kind/species of the probereagents present in the detection reagent can be the same. In otherembodiments, the detection reagent can include at least one differentkind/species of the probe reagents (e.g., 1, 2, 3, 4, 5, or 6 probereagent species). In such embodiments, the distinct probe reagentspecies can be different from the others by types (e.g., antibodies vs.DNA aptamers), binding domains, and/or target analytes.

Nucleic Acid Labels

In accordance with embodiments of various aspects described herein, thenucleic acid label or nucleic acid tag comprises at least onepre-determined nucleic acid subsequence, which is used to identify ananalyte or target. In some embodiments, the nucleic acid label ornucleic acid tag can comprise any number of the pre-determined nucleicacid subsequences, e.g., ranging from about 1 to about 100, from about 2to about 80, or from about 3 to about 50. In some embodiments, thenucleic acid label or nucleic acid tag can comprise at least about 1, atleast about 2, at least about 3, at least about 4, at least about 5, atleast about 6, at least about 7, at least about 8, at least about 9, atleast about 10, at least about 15, at least about 20, at least about 30,at least about 40, at least about 50, at least about 60, at least about70 or more pre-determined nucleic acid subsequences. In someembodiments, the nucleic acid label or nucleic acid tag can comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70 or morepre-determined nucleic acid subsequences. Without wishing to be bound,the minimum number of pre-determined nucleic acid subsequences (n)required in the detection reagent can vary upon the number of distinctprobes to be detected (X) and/or the number of distinct detectablelabels (e.g., optical labels such as fluorescent labels or quantum dots)available to be used (Y), and n can be determined by the equation:

${n = {{ceiling}\left( \frac{\ln\mspace{14mu} X}{\ln\mspace{14mu} Y} \right)}},$where the mathematical function “ceiling” refers to rounding up anon-integer number to the nearest integer, when needed. For example, if4 distinct detectable labels (Y) are used to distinguish 62 distinctprobe reagents (X), n=ceiling (2.98) ˜3. Therefore, at least threepre-determined nucleic acid subsequences are required in this example.

In some embodiments where the detectable labels include no-color (dark),the number of distinct detectable label available to be used can becomeY+1. In such embodiments, n can be determined by the equation:

${n = {{ceiling}\left( \frac{\ln\mspace{14mu} X}{\ln\left( {Y + 1} \right)} \right)}},$and thus fewer pre-determined nucleic acid subsequences can be used.However, the combination in which all readout cycles are dark should notbe allowed.

Further, the pre-determined nucleic acid subsequences can be designedsuch that each readout cycle can light up in multiple colors. Thus,2^(Y) instead of Y is used in the equation for n above and fewerpre-determined nucleic acid subsequences can thus be required. However,the combination in which all readout cycles are dark should not beallowed.

Additionally, while colors are generally used in a binary fashion, i.e.,whether the color is present or not, in the above examples, the colorintensity can also be used as a parameter of a signal signature. Forexample, if one detectable label is allowed to light up twice asbrightly in a different color than another, the multiplexing capacitycan be even further expanded.

The pre-determined nucleic acid subsequences can be constructed from anytypes of nucleic acids, including, but not limited to, DNA, RNA, PNA,LNA and any combinations thereof.

Each of the pre-determined subsequences of the nucleic acid label can beindependently of any length. In certain embodiments, the pre-determinedsubsequences can each independently comprise a length of about 1nucleobase to about 100 nucleobases, from about 1 nucleobase to about 50nucleobases, from about 2 nucleobases to about 50 nucleobases, fromabout 5 nucleobases to about 30 nucleobases, or from about 5 nucleobasesto about 20 nucleobases. In some embodiments, the pre-determinedsubsequences can each independently comprise one or more nucleobases,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or more nucleobases.

The achievable length of the pre-determined subsequences can affect thedegree of multiplexibility. In some embodiments, the achievable lengthof the pre-determined subsequences can be a function of the decreasingfluorescence intensity over many cycles of stripped and rehybridization(i.e. diffusion of the sequenceable particles, degradation, increasingbackground noise). In the case of DNA or RNA based nucleic acid labelsthat are amplified in situ, modified base is incorporated during itssynthesis (i.e., dUTP, biotin, Acrydite, aminoallele), enabling thesedetection reagents to be permanently embedded in a film (regardless ofthe film thickness) of functionalized polyacrylamide (i.e. Acryditestreptavidin, NHS ester Acrydite). The embedded material can then bestripped and rehybridized for many more cycles without altering itsspatial architecture and minimizing the background noise.

Two or more pre-determined subsequences can be conjugated togetherwithin a nucleic acid label using any methods known in the art. In someembodiments, two or more pre-determined subsequences can be conjugatedtogether by a sequence linker. The term “sequence linker” as used hereingenerally refers to an entity that connects two sequences orsubsequences as described herein together.

In some embodiments, the sequence linker can be a direct bond or an atomsuch as nitrogen, oxygen or sulfur; a unit such as NR₁, C(O), C(O)NH,SO, SO₂, SO₂NH; or a chain of atoms. If needed, the two ends of thepre-determined subsequences can be linked together by providing on thetwo ends of the pre-determined subsequences complementary chemicalfunctionalities that undergo a coupling reaction. In particularembodiments, the sequence linker is a direct bond, including, but notlimited to, a phosphodiester bond. For example, a 3′ carbon atom of asugar base at the 5′ end nucleotide of a first pre-determinedsubsequence can interact with the 5′ carbon atom of another sugar baseat the 3′ end nucleotide of a second pre-determined subsequence to forma covalent bond, e.g., a phosphodiester bond. As such, two or morepre-determined subsequences can be bonded together to form a longer andcontiguous pre-determined subsequence.

In some embodiments, the sequence linker can be a nucleotidic linker.The term “nucleotidic linker” as used herein refers to a linker of onenucleotide long or a sequence substantially comprising a plurality ofnucleotides. In some embodiments, the nucleotidic linker can have asequence length of at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20, at least 30 or more nucleotides. The sequencelength of the nucleotidic linker can vary with a number of factors,e.g., detection methods, and/or properties of optical labels. Withoutwishing to be bound by theory, in some embodiments, increasing thelength of the nucleotidic linker can increase the flexibility of thenucleic acid label, e.g., to increase the binding frequency between apre-determined sequence and a decoder probe, the term of which will bediscussed later. However, too long a nucleotidic linker can result in atoo long nucleic acid label, which could overlap with other nucleic acidlabels of the detection reagents during an assay and thus reduce thequality and/or accuracy of the signal detection. One of skill in the artcan determine the optimum length of the nucleotidic linker without undueexperimentations.

The nucleotidic linker can be in any structure or conformation. In someembodiments, the nucleotidic linker can be in a structure selected fromthe group consisting of single-stranded, double-stranded, partiallydouble-stranded, a hairpin, or any combinations thereof.

In some embodiments, the sequence linker can be a bead or a nanoparticleacting as a hub. Accordingly, two or more pre-determined subsequencescan be independently conjugated together via a bead or a nanoparticle.

Without wishing to be bound, while the sequence linker can be a directbond, an atom, a nucleotidic linker, or any combinations thereof, thesequence linker can also include a sequence of amino acids, a polymerchain, a microbead, a nanobead, or any combinations thereof. To providefor the linkages between sequence linker, in some embodiments, differentfunctionalities can be introduced to the ends of sequence linker and/orthe pre-determined subsequences. Examples of functionalities include,but are not limited to, amide groups, including carbonic acidderivatives, ethers, esters, including organic and inorganic esters,amino, urethane, urea and any combinations thereof.

In some embodiments, the nucleic acid label is substantially apolynucleotide sequence containing one or more pre-determinedsubsequences. In such embodiments, any two pre-determined subsequencesare either joined or conjugated together by a direct bond such as aphosphodiester bond (to produce longer contiguous subsequence), anucleotidic linker of any desirable length, or any combinations thereof.

In such embodiments, the nucleic acid label of the detection reagent canbe adapted to any configuration or structure. In some embodiments, thenucleic acid label can be single-stranded, double-stranded, partiallydouble-stranded, a hairpin, linear, circular, branched, a concatemer, orany combinations thereof. In some embodiments, the nucleic acid labelcan be a linear polynucleotide sequence.

In some embodiments, the nucleic acid label can be a circularpolynucleotide sequence. The advantage of using a circular nucleic acidlabel is that it can be amplified using rolling-circle amplification orhyperbranched rolling-circle amplification to generate a long continuousnucleic acid molecule that contains multiple copies of the same nucleicacid label sequences linked in series (also known as concatemer), thusresulting in an amplified signal. In some embodiments, instead ofpre-forming the detection reagents comprising the circular nucleic acidlabel(s) and the probe reagent(s), detection reagents comprising linearpolynucleotide(s) and the probe reagent(s) can be first synthesized. Thecircular nucleic acid labels can then added to hybridize with the linearpolynucleotide(s) before or after the probe reagent(s) bind to theanalytes. In such embodiments, while requiring an extra step, thisapproach can have the advantage over direct attachment of circularnucleic acid labels in that it does not require chemical modification ofthe nucleic acid label for conjugation with the probe reagents, thusresulting in a circular nucleic acid label that is compatible with abroader range of amplification enzymes. Furthermore, the linearpolynucleotides can be smaller than the secondary circular nucleic acidlabels, facilitating diffusion of the probe reagents (and the detectionreagents) to their targets. In other embodiments, the linearpolynucleotides can be circularized using a suitable double-stranded orsingle-stranded ligase, with or without the addition of a suitableligation template. Without wishing to be bound by theory, suchembodiments can avoid the extra hybridization that is required when apre-circularized oligonucleotide is used instead.

When the detection reagents described herein are used as FISH probes,the nucleic acid labels and the FISH probes can be part of the samenucleic acid sequence construct, and thus the circularization canencompass the entire construct (e.g., both the nucleic acid labels andFISH probes). A schematic representation of exemplary FISH probes forSeqTagged FISH is shown in FIG. 13.

In some embodiments, the method described herein can be used to identifya class an analyte, e.g., a pathogen belongs to. For example, the methodcan be used to identify if a pathogen is a Gram-negative, Gram-positive,or some other class of pathogen, e.g., yeast. Thus, the method describedherein can be used to as Gram test to determine whether a suspectedpathogen is Gram positive, Gram negative or yeast. This can be usefulfor quickly identifying the type of infection in a subject andadministering appropriate therapy. By way of example only, this can beaccomplished using a detection reagent comprising a class-specificprobe, also referred to as “Gram-stain like probe” herein. Again by wayof example only, the probe can be a DNA probe for FISH.

In some embodiment, the FISH probe for identifying eubacteria cancomprise the nucleotide sequence of SEQ ID NO: 4 (GCTGCCTCCCGTAGGAGT).An exemplary SeqTag labeled FISH-probe for identifying eubacteria cancomprise the nucleotide sequence of SEQ ID NO: 5(CTGCCTCCCGTAGGAGTTTTTTCGCTTTAGCCTAAGTGAAATC).

In some embodiments, the FISH probe for identifying yeast can comprisethe nucleotide sequence of SEQ ID NO: 6 (CTCTGGCTTCACCCTATTC. Anexemplary SeqTag labeled FISH-probe for identifying yeast can comprisethe nucleotide sequence of SEQ ID NO: 7(CTCTGGCTTCACCCTATTCTTTTTCGCTTTTTTGGGGAAAAGACA).

In some embodiments, the FISH probe for identifying firmicutes cancomprise the nucleotide sequence of SEQ ID NO: 8 (CGGAAGATTCCCTACTGC).An exemplary SeqTag labeled FISH-probe for identifying yeast cancomprise the nucleotide sequence of SEQ ID NO: 9(CGGAAGATTCCCTACTGCTTTTTCGCTTTCTGTAATGGAGTGGA).

In the SeqTag labeled FISH probes discussed above, each probe can beassigned a particular “signal signature” for each of the three readoutsteps. For example, colors can be labeled at B, C, and D. In someembodiments, each color can be from a different fluorophore, such asFAM, Cy3 and Cy5. Each signal signature can take advantage of multiplefluorophores simultaneously or even the same fluorophore multiple times,e.g., a SeqTag with signal signature corresponding to “DDDC.”

An exemplary signal signature for identifying eubacteria, yeast orfirmicutes is shown in Table 1. As shown, each class has a differentassigned code for the first readout step. For example, for the firstreadout, eubacteria are assigned color C′ yeast color D and firmicutescolor B. On readout, eubacteria will show up as color C, yeast as colorD, firmicutes as both colors B and C.

TABLE 1 Assigned code Effective code Name S1 S2 S3 S1 S2 S3Eubacteria-Kempf C C All_yeast-Kempf D D Firmicutes-pB-00196 B BC

After identifying the class of pathogens, the pathogens can be furtherprobed for identifying the specific pathogen or genus using the methoddescribed herein. For example, the detection reagent can comprise apathogen specific probe that binds to a specific pathogen or genus ofpathogens. For example, the probe can be a FISH probe that specificallybinds to a specific pathogen. Some exemplary pathogen specific FISHprobes are shown in Table 2.

TABLE 2 SEQ ID NO: Name probeBase FISH Sequence Assigned codeEffective code Gram + 10 Staph_spp-Kempf TCCTCCATATCTCTGCGC DD B BC DD B11 S_Aureus-Kempf GAAGCAAGCTTCTCGTCCG CD B BC CDDD BB 12Streptococcus_spp- CACTCTCCCCTTCTGCAC DD D BC DD D Kempf 13S_Pneumoniae- GTGATGCAAGTGCACCTT B DC BC DDB DDC Kempf 14 S_Pyogenes-TTCCAAAGCGTACATTGGTT C DB BC DDC DDB Kempf 15 S_Agalactiae-GTAAACACCAAACMTCAGCG D DC BC DDD DDC Kempf 16 B_subtilis-pB-CGA AGG GGA CGT CCT ATC T C DD BC C DD 00401 17 L_acidophilus-pB-CAG GCT TGC TCC TCG TTG DD C BC DD C 00711 Gram − 18 P_Aeruginosa-TCTCGGCCTTGAAACCCC B B C B B Kempf 19 K_Pneumoniae- CCTACACACCAGCGTGCC BDD C B DD Kempf 20 H_influenzae-pB- CCG CAC TTT CAT CTT CCG B BC C B BC00348 21 B_cepacia-pB- CTG TGC GCC GGT TCT CTT DD B C DD B 00346 22K_oxytoca-pB- CTA CAA GAC TCC AGC CTG CC DD C C DD C 01681 23E_coli-pB-02569- ATG AGC AAA GGT ATT AAC TTT ACT B C C B C compl CCC 24Shewanella_spp- AGC TAA TCC CAC CTA GGT TCA TC BC B C BC B pB-01191-mod 25 H_pylori-pB-00361 CACACCTGACTGACTATCCCG C B C C B Yeast 26C_Albicans-Kempf GCCAAGGCTTATACTCGCT C BC D C BC 27 C_Glabrata_KempfCCGCCAAGCCACAAGGACT C CD D C CD 28 C_Krusei-Kempf GATTCTCGGCCCCATGGG BCC D BC C 29 C_Parapsilosis- CCTGGTTCGCCAAAAAGGC CD C D CD C Kempf

Exemplary SeqTag labeled FISH-probe for identifying a specific pathogenare shown in Table 3. As shown in Table 3, each specific pathogen can beassigned a specific signal signature based on an assigned color for eachreadout step and identity of a pathogen can be decoded using this tablewhen the method is carried out using this set of assigned coding.

TABLE 3 SEQ ID NO: Name SeqTag-labeled FISH-probe Sequence Gram + 30Staph_spp-Kempf TCCTCCATATCTCTGCGCTTTTTCGCTTTCTGGAGAAAGGGCCATTTTTCGCTTTCGGTTCCAAAGACACTTTTTCGCTTTCTGGAGAAAGGGCC A 31 S_Aureus-KempfGAAGCAAGCTTCTCGTCCGTTTTTCGCTTTCTGGAGAAAGGGCCATTTTTCGCTTTCGGTTCCAAAGACACTTTTTCGCTTTGGAAGCACCTAT TCC 32Streptococcus_spp- CACTCTCCCCTTCTGCACTTTTTCGCTTTCTGGAGAAAGGGCCATTTTKempf TCGCTTTTCACGATCCCATGTATTTTTCGCTTTCTGGAGAAAGGGCC A 33S_Pneumoniae-Kempf GTGATGCAAGTGCACCTTTTTTTCGCTTTGAAGCCGGTTATAGCTTTTTCGCTTTTAGGCATTAGCATTGTTTTTCGCTTTTCACGATCCCATGT A 34 S_Pyogenes-KempfTTCCAAAGCGTACATTGGTTTTTTTCGCTTTCGGTTCCAAAGACACTTTTTCGCTTTGGAAGCACCTATTCCTTTTTCGCTTTTCACGATCCCAT GTA 35S_Agalactiae-Kempf GTAAACACCAAACMTCAGCGTTTTTCGCTTTGAAGCCGGTTATAGCTTTTTCGCTTTCTGGAGAAAGGGCCATTTTTCGCTTTTCACGATCCC ATGTA 36B_subtilis-pB-00401 CGA AGG GGA CGT CCT ATCTTTTTTCGCTTTTCACGATCCCATGTATTTTTCGCTTTGGAAGCACCTATTCCTTTTTCGCTTTTCACGATCCCATGTA 37 L_acidophilus-pB- CAG GCT TGC TCC TCG00711 TTGTTTTTCGCTTTCTGGAGAAAGGGCCATTTTTCGCTTTGAAGCCGGTTATAGCTTTTTCGCTTTCTGGAGAAAGGGCCA Gram − 38 P_Aeruginosa-KempfTCTCGGCCTTGAAACCCCTTTTTCGCTTTTAGGCATTAGCATTGTTTT TCGCTTTCGGTTCCAAAGACAC39 K_Pneumoniae-Kempf CCTACACACCAGCGTGCCTTTTTCGCTTTTCACGATCCCATGTATTTTTCGCTTTTAGGCATTAGCATTGTTTTTCGCTTTTCACGATCCCATGTA 40 H_influenzae-pB-CCG CAC TTT CAT CTT 00348CCGTTTTTCGCTTTCGGTTCCAAAGACACTTTTTCGCTTTTAGGCATTAGCATTGTTTTTCGCTTTGAAGCCGGTTATAGC 41 B_cepacia-pB-00346CTG TGC GCC GGT TCT CTTTTTTTCGCTTTCTGGAGAAAGGGCCATTTTTCGCTTTCGGTTCCAAAGACACTTTTTCGCTTTCTGGAGAAAGGGCCA 42 K_oxytoca-pB-01681CTA CAA GAC TCC AGC CTG CCTTTTTCGCTTTCTGGAGAAAGGGCCATTTTTCGCTTTGAAGCCGGTTATAGCTTTTTCGCTTTCTGGAGAAAGGGCCA 43 E_coli-pB-02569-ATG AGC AAA GGT ATT AAC TTT ACT complCCCTTTTTCGCTTTTAGGCATTAGCATTGTTTTTCGCTTTGAAGCCGG TTATAGC 44Shewanella_spp-pB- AGC TAA TCC CAC CTA GGT TCA 01191-modTCTTTTTCGCTTTTAGGCATTAGCATTGTTTTTCGCTTTCGGTTCCAAAGACACTTTTTCGCTTTGGAAGCACCTATTCC 45 H_pylori-pB-00361CACACCTGACTGACTATCCCGTTTTTCGCTTTGGAAGCACCTATTCCTTTTTCGCTTTCGGTTCCAAAGACAC Yeast 46 C_Albicans-KempfGCCAAGGCTTATACTCGCTTTTTTCGCTTTCGGTTCCAAAGACACTTTTTCGCTTTGGAAGCACCTATTCCTTTTTCGCTTTGAAGCCGGTTATA GC 47 C_Glabrata_KempfCCGCCAAGCCACAAGGACTTTTTTCGCTTTGAAGCCGGTTATAGCTTTTTCGCTTTGGAAGCACCTATTCCTTTTTCGCTTTTCACGATCCCATG TA 48 C_Krusei-KempfGATTCTCGGCCCCATGGGTTTTTCGCTTTTAGGCATTAGCATTGTTTTTCGCTTTGAAGCCGGTTATAGCTTTTTCGCTTTGGAAGCACCTATTC C 49C_Parapsilosis-Kempf CCTGGTTCGCCAAAAAGGCTTTTTCGCTTTCTGGAGAAAGGGCCATTTTTCGCTTTGAAGCCGGTTATAGCTTTTTCGCTTTGGAAGCACCTAT TCC

As shown in Table 3, the “signal signature” can comprise multiplefluorescence “colors” in each step. For example, E. coli can be markedas red in step 1 and red+green in step 2 and blue in step 3. The “signalsignature” can also be encoded in the brightness of the color at eachstep. For example, E. coli could be marked as red in step 1 and 3 timesbrighter red in step 2.

Exemplary hybridization sites for generating the different signalsignatures at different steps using the exemplary SeqTag labeledFISH-probes described above are shown in Table 4.

TABLE 4 SEQ ID NO: Assigned “Color” Set1 50 B CGCTTTCTGTAATGGAGTGGA 51 CCGCTTTAGCCTAAGTGAAATC 52 D CGCTTTTTTGGGGAAAAGACA Set 2 53 BCGCTTTTAGGCATTAGCATTG 54 C CGCTTTGGAAGCACCTATTCC 55 DCGCTTTCTGGAGAAAGGGCCA Set 3 56 B CGCTTTCGGTTCCAAAGACAC 57 CCGCTTTGAAGCCGGTTATAGC 58 D CGCTTTTCACGATCCCATGTA

In some embodiments, multiple detection reagents can be used to identifya specific analyte. For example, a first set of probes can be used toidentify whether the potential pathogen is gram-position, gram-negativeand then another set of probes to identify it more specifically. Bothprobes would need to produce a detectable signal, allowing one to use,for example, Table 3 to uniquely identify the pathogen.

In some embodiments, the nucleic acid label can be a concatemer,including a rolony or a DNA nanoball that is large enough to act as aparticle rather than simply a strand of nucleic acid. Using a concatemeras a nucleic acid label can eliminate the need for in-situ enzymatictreatment of circular nucleic acid amplification, but it can alsoincrease the overall molecular weight of the detection reagent and thusretard diffusion. Similar to the circular nucleic acid labels asdescribed above, an exemplary approach of hybridizing concatemers withthe linear polypeptides of the detection reagents after probe reagentsbind to the analytes can be used to facilitate diffusion of the probereagents (and the detection reagents) to their targets.

To increase the accuracy and/or specificity of the methods describedherein, in various embodiments, the nucleic acid label can be designedfor minimal cross-hybridization of bases with each other. Variousart-recognized computational programs or algorithms are available todesign nucleic acid sequences with minimal cross-hybridization. Thus,one of skill in the art can optimize the nucleic acid label sequenceusing any methods or algorithms known in the art.

In some embodiments, the nucleic acid labels described herein can besynthetic nucleic acid molecules (e.g., DNA, RNA, or DNA/RNA hybrids),and can be rationally-designed to have features that optimize labelingand detection of the detection reagents, and that prevent secondarystructure formation. In some embodiments, a nucleic acid label is adesigned polynucleotide sequence from about 50 to 50,000 bases long.

In some embodiments, the nucleic acid labels described herein can bedesigned to minimize predictable secondary structures, and/or bedesigned such that each nucleic acid label can hybridize only againstits own target. In some embodiments, the nucleic acid labels describedherein can be designed to be devoid of any secondary structure. Putativesecondary structures (e.g. hairpins, folding, or internal base pairing)can be predicted by methods known in the art such as MFOLD. Withoutintending to be limited to any theory, in some embodiments, predictablesecondary structure in the nucleic acid label can be minimized byavoiding inverted repeats and by skewing the backbone-specific contentsuch that the backbone of the nucleic acid label is CT or GA-rich. Anyart-recognized methods, e.g., MFOLD, can be used to verify if eachnucleic acid label can hybridize only against its own target.

Sequences can also be screened to avoid common six-base-cutterrestriction enzyme recognition sites. Selected sequences can beadditionally subjected to predicted secondary structure analysis, andthose with the least secondary structure may be chosen for furtherevaluation. Any program known in the art can be used to predictsecondary structure, such as the MFOLD program (Zuker, 2003, NucleicAcids Res. 31 (13):3406-15; Mathews et al., 1999, J. Mol. Biol.288:911-940).

In some embodiments, the nucleic acid label can comprise only a subsetof the A, G, C, T (and/or U) nucleotides or modified nucleotidesthereof. In some embodiments, the nucleic acid label can comprise onlyone of the A, G, C, T (and/or U) nucleotides or modified nucleotidesthereof. In some embodiments, the nucleic acid can comprise two of theA, C, T (and/or U) nucleotides or modified nucleotides thereof. In someembodiments, the nucleic acid label can comprise only three of the A, G,C, and T (and/or U) nucleotides or modified nucleotides thereof. In someembodiments, the nucleic acid label can comprise only four of the A, G,C, and T (and/or U) nucleotides or modified nucleotides thereof. Thesubset of the A, G, C and T (and/or U) nucleotides or modifiednucleotide thereof can be selected from the group consisting of A; G; C;T; U; (A,G); (A,C); (G,T); (G,U); (C,T); (C,U); (G,T,U); (C,T,U);(A,C,G); (A,G,T); (A,G,U); (A,C,T); (A,C,U); (C,G,T); (C,G,U);(A,C,T,U); and (C,G,T,U).

Without wishing to be bound, the nucleic acid label can be a modifiednucleic acid label. An exemplary modification of the nucleic acid labelincludes, without limitations, attaching one or more detectablemolecules to the nucleic acid label (either to one end of or along thenucleic acid label sequence). The detectable molecule can be any opticalmolecule, including, but not limited to, a small-molecule dye, afluorescent protein, a quantum dot, or any combinations thereof. Inanother embodiment, at least one end of the nucleic acid label can bemodified to include a chemical functional group and/or a protein orpeptide to facilitate the conjugation between the nucleic acid label andthe probe reagent.

In some embodiments, at least one (including, e.g., at least two, atleast three, at least four, at least five, at least six or more) nucleicacids or nucleotides present in the nucleic acid label can be modified.For example, the nucleic acid can comprise one or more nucleic acidmodifications as described herein, e.g., selected from the groupconsisting of internucleotide linkage modifications (intersugar linkagemodifications), sugar modifications, nucleobase modifications, backbonemodifications/replacements, and any combinations thereof.

Conjugation between a Nucleic Acid Label and a Probe Reagent

In accordance with embodiments of various aspects described herein, thedetection reagent comprises at least one probe reagent and at least onenucleic acid label, wherein the probe reagent and the nucleic acid labelcan be conjugated together by any methods known in the art.

In some embodiments, the probe reagent and the nucleic acid label can beattached or conjugated together by a linker. As used herein, the term“linker” generally refers to an entity that connects the probe reagentand the nucleic acid label together. The linker can be monovalent ormultivalent. The term “monovalent” as used herein refers to the capacityof a linker to join one probe reagent to one nucleic acid label. Theterm “multivalent” as used herein refers to the capacity of a linker tobind with one or more probe reagents and/or nucleic acid labels. In someembodiments, a multivalent linker can join at least one probe reagent toa plurality of nucleic acid labels (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore nucleic acid labels).

In some embodiments, the term “linker” means an organic moiety thatconnects two parts of a compound. Such linkers typically comprise adirect bond or an atom such as oxygen or sulfur, a unit such as NH,C(O), C(O)NH, SO, SO₂, SO₂NH, SS, or a chain of atoms, such assubstituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedC₂-C₆ alkenyl, substituted or unsubstituted C₂-C₆ alkynyl, substitutedor unsubstituted C₆-C₁₂ aryl, substituted or unsubstituted C₅-C₁₂heteroaryl, substituted or unsubstituted C₅-C₁₂ heterocyclyl,substituted or unsubstituted C₃-C₁₂ cycloalkyl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, NH,C(O).

In some embodiments, the linker is a branched linker. The branchpoint ofthe branched linker may be at least trivalent, but can be a tetravalent,pentavalent or hexavalent atom, or a group presenting such multiplevalencies. In some embodiments, the branchpoint is —N, —N(R)—C, —O—C,—S—C, —SS—C, —C(O)N(R)—C, —OC(O)N(R)—C, —N(R)C(O)—C, or —N(R)C(O)O—C;wherein R is independently for each occurrence H or optionallysubstituted alkyl. In some embodiments, the branchpoint is glycerol orderivative thereof.

In some embodiments, linker comprises a cleavable linking group. As usedherein, a “cleavable linking group” is a chemical moiety which issufficiently stable outside the cell, but which upon entry into a targetcell is cleaved to release the two parts the linker is holding together.In a preferred embodiment, the cleavable linking group is cleaved atleast 10 times or more, preferably at least 100 times faster in thetarget cell or under a first reference condition (which can, e.g., beselected to mimic or represent intracellular conditions) than in theblood or serum of a subject, or under a second reference condition(which can, e.g., be selected to mimic or represent conditions found inthe blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; amidases; endosomes or agents that cancreate an acidic environment, e.g., those that result in a pH of five orlower; enzymes that can hydrolyze or degrade an acid cleavable linkinggroup by acting as a general acid, peptidases (which can be substratespecific) and proteases, and phosphatases.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, for livertargeting, cleavable linking groups can include an ester group. Livercells are rich in esterases, and therefore the linker will be cleavedmore efficiently in liver cells than in cell types that are notesterase-rich. Other cell-types rich in esterases include cells of thelung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In some embodiments, cleavable linking group is cleaved at least 1.25,1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in the cell (orunder in vitro conditions selected to mimic intracellular conditions) ascompared to blood or serum (or under in vitro conditions selected tomimic extracellular conditions). In some embodiments, the cleavablelinking group is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 10%, 5%, or 1% in the blood (or in vitro conditions selected tomimic extracellular conditions) as compared to in the cell (or under invitro conditions selected to mimic intracellular conditions).

Exemplary cleavable linking groups include, but are not limited to,redox cleavable linking groups (e.g., —S—S— and —C(R)₂—S—S—, wherein Ris H or C₁-C₆ alkyl and at least one R is C₁-C₆ alkyl such as CH₃ orCH₂CH₃); phosphate-based cleavable linking groups (e.g., —O—P(O)(OR)—O—,—O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—, —O—P(O)(OR)—S—,—S—P(O)(OR)—S—, —O—P(S)(ORk)-S—, —S—P(S)(OR)—O—, —O—P(O)(R)—O—,—O—P(S)(R)—O—, —S—P(O)(R)—O—, —S—P(S)(R)—O—, —S—P(O)(R)—S—,—O—P(S)(R)—S—, —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—,—S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—,P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—,—S—P(O)(H)—S—, and —O—P(S)(H)—S—, wherein R is optionally substitutedlinear or branched C₁-C₁₀ alkyl); acid celavable linking groups (e.g.,hydrazones, esters, and esters of amino acids, —C═NN— and —OC(O)—);ester-based cleavable linking groups (e.g., —C(O)O—); peptide-basedcleavable linking groups, (e.g., linking groups that are cleaved byenzymes such as peptidases and proteases in cells, e.g.,—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids). A peptide based cleavable linking groupcomprises two or more amino acids. In some embodiments, thepeptide-based cleavage linkage comprises the amino acid sequence that isthe substrate for a peptidase or a protease found in cells.

In some embodiments, an acid cleavable linking group is cleaveable in anacidic environment with a pH of about 6.5 or lower (e.g., about 6.0,5.5, 5.0, or lower), or by agents such as enzymes that can act as ageneral acid.

In addition to covalent linkages, two parts of a compound can be linkedtogether by an affinity binding pair. The term “affinity binding pair”or “binding pair” refers to first and second molecules that specificallybind to each other. One member of the binding pair is conjugated withfirst part to be linked while the second member is conjugated with thesecond part to be linked. As used herein, the term “specific binding”refers to binding of the first member of the binding pair to the secondmember of the binding pair with greater affinity and specificity than toother molecules.

Exemplary binding pairs include any haptenic or antigenic compound incombination with a corresponding antibody or binding portion or fragmentthereof (e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulinand goat antimouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin, hormone[e.g., thyroxine and cortisol-hormone binding protein, receptor-receptoragonist, receptor-receptor antagonist (e.g., acetylcholinereceptor-acetylcholine or an analog thereof), IgG-protein A, IgG-proteinG, IgG-synthesized protein AG, lectin-carbohydrate, enzyme-enzymecofactor, enzyme-enzyme inhibitor, and complementary oligonucleotidepairs capable of forming nucleic acid duplexes), and the like. Thebinding pair can also include a first molecule which is negativelycharged and a second molecule which is positively charged.

One example of using binding pair conjugation is the biotin-avidin,biotin-streptavidin or biotin-neutravidin conjugation. In this approach,one of the molecule or the peptide is biotinylated and the other isconjugated with avidin or streptavidin. Many commercial kits are alsoavailable for biotinylating molecules, such as proteins.

Another example of using binding pair conjugation is the biotin-sandwichmethod. See, e.g., example Davis et al., Proc. Natl. Acad. Sci. USA,103: 8155-60 (2006). The two molecules to be conjugated together arebiotinylated and then conjugated together using at least one tetravalentavidin-like molecule (e.g., avidin, streptavidin, or neutravidin) as alinker.

Accordingly, in some embodiments, both the nucleic acid label(s) andprobe reagent(s) can be biotinylated and then linked together using anavidin-like molecule (e.g., avidin, streptavidin, or neutravidin). Inone embodiment, neutravidin and/or streptavidin is used as a linker tobridge together the biotinylated nucleic acid label(s), e.g., DNAsequence(s), and biotinylated probe reagent(s), e.g., antibody. Withoutwishing to be bound by theory, each avidin-like molecule (e.g., avidin,streptavidin, or neutravidin) generally has four binding sites, so atmost four molecules can be linked together. For example, onebiotinylated probe reagent can be linked, via an avidin-like molecule(e.g., avidin, streptavidin or neutravidin), to three biotinylatednucleic acid labels, or in any other combinations (e.g., twobiotinylated probe reagents linked to two biotinylated nucleic acidlabels).

Biotinylation of a nucleic acid label (i.e., attaching a biotin to anucleic acid label) can occur at any location of the nucleic acid label.In some embodiments, the nucleic acid label can by synthesized ormodified with a terminal biotin, i.e., the nucleic acid label can have abiotin at its 5′ end and/or 3′ end. In other embodiments, the nucleicacid label can by synthesized or modified with an internal biotin, i.e.,the nucleic acid label can have a biotin anywhere between its 5′ and 3′ends, but not at its 5′ and/or 3′ ends. Such internal biotinylation canleave at least one end (e.g., both ends) of the nucleic acid labelaccessible, allowing the nucleic acid label to be circularized after thenucleic acid label has bound, which can in turn, for example, enablerolling circle amplification as described earlier. Chemical modificationof the nucleic acid label, e.g., to attach a terminal and/or an internalbiotin to the nucleic acid label after synthesis, is within one of skillin the art.

For some embodiments where the probe reagent is a nucleic acid, anexample of using binding pair conjugation is double-stranded nucleicacid conjugation. In this approach, the first part to be linked isconjugated is with linked a first strand first strand of thedouble-stranded nucleic acid and the second part to be linked isconjugated with the second strand of the double-stranded nucleic acid.Nucleic acids can include, without limitation, defined sequence segmentsand sequences comprising nucleotides, ribonucleotides,deoxyribonucleotides, nucleotide analogs, modified nucleotides andnucleotides comprising backbone modifications, branchpoints andnonnucleotide residues, groups or bridges.

In some embodiments, the linker can be a linker molecule. Examples oflinker molecules can include, but are not limited to, a polymer, sugar,nucleic acid, peptide, protein, hydrocarbon, lipid, polyethelyne glycol,crosslinker, or combination thereof.

Non-limiting examples of crosslinkers that can be used as linkermolecules can include, but are not limited to, amine-to-aminecrosslinkers (e.g., but are not limited to the ones based on NHS-esterand/or imidoester reactive groups), amine-to-sulfhydryl crosslinkers,carboxyl-to-amine crosslinkers (e.g., but are not limited to,carbodiimide crosslinking agents such as DCC, and/or EDC (EDAC); and/orN-hydroxysuccinimide (NHS)), photoreactive crosslinkers (e.g., but notlimited to, aryl azide, diazirine and any art-recognized photo-reactive(light-activated) chemical crosslinking reagents),sulfhydryl-to-carbohydrate crosslinkers (e.g., but are not limited tothe ones based on malemide and/or hydrazide reactive groups),sulfhydryl-to hydroxyl crosslinkers (e.g., but are not limited to theones based on maleimide and/or isocyanate reactive groups),sulfhydryl-to-sulfhydryl crosslinkers (e.g., but are not limited to,maleimide and/or pyridyldithiol reactive groups), sulfo-SMCCcrosslinkers, sulfo-SBED biotin label transfer reagents,sulfhydryl-based biotin label transfer reagents, photoreactive aminoacids (e.g., but are not limited to diazirine analogs of leucine and/ormethionine), NHS-azide Staudinger ligation reagents (e.g., but are notlimited to, activated azido compounds), NHS-phosphine Staudingerligation reagents (e.g., but are not limited to, activated phosphinecompounds), and any combinations thereof.

In some embodiments, any commercially available crosslinkers (e.g., butnot limited to the ones from Thermo Scientific or Piercenet, Rockford,Ill.) can be used as a linker molecule herein.

In some embodiments, the term “linker” as used herein can be a physicalsubstrate. In some embodiments, the physical substrate is a particle.The particle can be of any shape, e.g., spheres; rods; shells; beads,tubes, wires, and prisms; and these particles can be part of a network.

The particles can be made of any materials. In some embodiments, theparticle can comprise a material selected from the group consisting ofmetal (e.g., gold, or iron), metal oxides (e.g., iron oxide), plastic,glass, polymer (e.g., polystyrene), and any combinations thereof.

For in vivo purposes, e.g., disease and/or pathogen diagnosis and/ortargeted drug delivery, the polymer can be biocompatible and/orbiodegradable. The term “biocompatible polymer” refers to polymerswhich, in the amounts employed, are non-toxic and substantiallynon-immunogenic when used internally in the patient and which aresubstantially insoluble in the body fluid of the mammal. Examples ofnon-biodegradable biocompatible polymers include, by way of example,cellulose acetates (including cellulose diacetate), ethylene vinylalcohol copolymers, hydrogels (e.g., acrylics), polyacrylonitrile,polyvinylacetate, cellulose acetate butyrate, nitrocellulose, copolymersof urethane/carbonate, copolymers of styrene/maleic acid, and anycombinations thereof. Biodegradable polymers are known in the art, e.g.,without limitations, linear-chain polymers such as polylactides,polyglycolides, polycaprolactones, polyanhydrides, polyamides,polyurethanes, polyesteramides, polyorthoesters, polydioxanones,polyacetals, polyketals, polycarbonates, polyorthocarbonates,polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),poly(amino acids), polyvinylpyrrolidone, polyethylene glycol,polyhydroxycellulose, chitin, chitosa, and copolymers, terpolymers andany combinations thereof. Other biodegradable polymers include, forexample, fibrin, gelatin, collagen, and any combinations thereof.

The particles can be of any size for the purpose of various aspectsdescribed herein, provided that the particle size does not significantlyaffect the diffusion property of the detection reagent. For example, theparticle size can range from 5 nm to 1 mm, from about 10 nm to about 500μm, or from about 50 nm to about 250 μm. In some embodiments, a particledescribed herein is a nanoparticle or a microparticle. As used herein,the term “nanoparticle” refers to particles that are on the order of10⁻⁹ or one billionth of a meter and below 10⁻⁶ or 1 millionth of ameter in size. The term “microparticle” as used herein refers toparticles that are on the order of 10⁻⁶ or one millionth of a meter andbelow 10⁻³ or 1 thousandth of a meter in size. In some embodiments, theparticle can be selected from a group consisting of a gold nanoparticleor microparticle, a paramagnetic nanoparticle or microparticle, amagnetic nanoparticle or microparticle, a polystyrene nanoparticle ormicroparticle, a nanotube or a microtube, a nanowire or a microwire, andany combinations thereof.

The particles can be adapted to possess at least one additionalproperty, depending on various applications. In some embodiments, theparticles can be adapted to be magnetic responsive orparamagnetic-responsive, e.g., magnetic or paramagnetic particles. Insome embodiments, the particles can be adapted to be a delivery vehicle.For example, in some embodiments, the particles can be encapsulated witha therapeutic agent, e.g., for targeted drug delivery to treat a diseaseor disorder.

In some embodiments, the particles can be modified. For example, theparticles can be conjugated with proteins, peptides, nucleic acids, orany combinations thereof. In one embodiment, the particles can besurface-conjugated or coated with one member of the binding pair asdescribed above (e.g., for biotin-streptavidin interaction,streptavidin-coated particles can be used for the purpose of variousaspects described herein). In some embodiments, the particles can besurface-activated with functional groups (e.g., but not limited to,amine, carboxylic acid, epoxy, tosyl, silane, and any combinationsthereof), e.g., to provide binding sites for probe reagents and/ornucleic acid labels. Methods for surface modifications of particles,such as nanoparticles or microparticles, are well known in the art. Oneof skill in the art can readily modify or activate the surface ofparticles using any art-recognized reactions, e.g., covalently throughcrosslinkers, or through protein interaction.

The particles described herein can be used in conjunction with theorganic linker described above, to form a conjugate linker, or theparticles can be used alone as a linker. Both the nucleic acid labelsand the probe reagents can be coupled to the particles using a multitudeof methods. These include, but are not limited to, direct covalentattachment such as using chemical crosslinkers based on chemistries suchas NHS, maleimide, tosyl, isocyanide, etc.; chemical linkage such asusing EDC chemistry, thiol adsorption to gold, vinyl/acrylate radicalreaction, acrylate-based addition (of e.g., thiols); and proteinmediated couplings based on proteins such as streptavidin (and itsrelatives such as avidin, neutravidin, etc.), protein A, and secondaryantibodies, and any combinations thereof.

The particles can act as hubs that facilitate a conjugation of multiplenucleic acid labels to single or multiple probes. Depending onapplications and/or properties of nucleic acid labels and/or probereagents, particles can be used as hubs for multi-conjugation. Forexample, in some embodiments, by acting as a hub, the particles canallow multiple nucleic acid labels to be present at each location (e.g.,location of a target molecule or analyte) where the probe binds.Accordingly, the signal generated from the detection reagent can beamplified and thus greater than if only a single label was present. Thisis especially desirable where antigens/targets or analytes are sparse ina sample.

In some embodiments, the particles can allow multiple probes to bearranged in proximity to each other. Thus, the particles can allowseveral weaker binding events to combine into strong binding. This“avidity action” can transform probe reagents with individually weakaffinities into effective sensors.

In some embodiments, by acting as a hub, the particles can allow eachprobe reagent to conjugate to multiple nucleic acid labels. Thiscapability can eliminate the need for other amplification methods (e.g.,rolling circle amplification) as multiple nucleic acid labelscorresponding to a probe reagent can be used as a form of signalamplification. In some embodiments, this capability can also be used toseparate the detection reagents with one nucleic acid label from theones with different nucleic acid labels, which can, in turn, enable, forexample, sequencing by single-base extension. Furthermore, differentnucleic acid labels can be conjugated to the particles at controlledratios, and the ratios can encode additional information. By way ofexample only, to capture in situ information of one or more enzymes'behavior (e.g., relative enzyme concentration) in the presence of ananalyte, two or more different nucleic acid labels can be conjugated tothe particles, wherein a subpopulation of the nucleic acid labelscorresponding to the probe reagent is not cleavable, while othersubpopulations of the nucleic acid labels are each adapted to becleavable in the presence of their respective enzymes. In suchembodiments, the cleavable nucleic acid labels can be conjugated to theparticles by cleavable peptide bonds specific for corresponding enzymes.By comparing the ratios of signals generated from the various nucleicacid labels, one would be able to determine the relative enzymeconcentrations in the presence of an analyte.

The arrangement of the probe reagent(s) and/or nucleic acid label(s) onthe particles can vary with a number of factors, e.g., applications,properties of probe reagents and/or nucleic acid labels, sampleproperties, and/or analytes of interests. In some embodiments, the probereagents and nucleic acid labels can be conjugated to the particledirectly (see, e.g., FIG. 4A). In some embodiments, one component can belinked to the particle through the other. In such embodiments, the probereagents can be conjugated or linked to the particle through the nucleicacid labels (see, e.g., FIG. 4B), e.g., to allow the probe reagentsbeing more accessible by the corresponding analytes in a sample.

In some embodiments, the detection reagents can further comprise atleast one substrate linker conjugated to the particle. In suchembodiment, the substrate linker can allow the detection reagents to beimmobilized to a solid support or substrate.

Detectable Molecules or Detectable Labels

A detectable molecule or detectable label can be covalently attached toa decoder probe or complementary nucleobase before or after the decoderprobe or the complementary nucleobase is attached to the pre-determinedsubsequences or hybridization sites of a nucleic acid label. Forexample, in attaching a detectable label to a decoder probe, the labelcan be covalently attached by incorporation of a nucleotide containing adetectable label into the nucleic acid during its synthesis, but beforeit is hybridized the pre-determined subsequence of the nucleic acidlabel. Alternatively, during the synthesis of a decoder probe sequence,a nucleotide containing a detectable label acceptor group can beincluded, and the detectable label can be added to the decoder probeafter its synthesis, either before or after it is hybridized to thepre-determined subsequences of the nucleic acid label. Alternatively,the detectable label can be indirectly attached to the decoder probe,for example, by incorporating a nucleotide containing a ligand-bindingmolecule (e.g., biotin) into the decoder probe during synthesis, and byadding a ligand (e.g., streptavidin) that is covalently attached to thedetectable molecule, or vice versa.

In some embodiments, the ratios of a detectable label to a decoder probecan range from about 1:1 to about 100:1, from 1:1 to about 50:1, fromabout 1:1 to about 25:1, from about 1:1 to about 10:1, or from about 1:1to about 5:1. When a decoder probe comprises more than one detectablelabel, each detectable label can be attached to a nucleotide of thedecoder probe.

A detectable label or a detectable molecule can be attached to anynucleotide including both natural and non-natural nucleotides. Anucleotide contains three parts, a phosphate group, a pentosefive-carbon sugar molecule, and an organic base. In RNA, the pentose isribose and in DNA it is deoxyribose and so nucleotides for incorporationinto RNA are called ribonucleotides and nucleotides for incorporationinto DNA are called deoxyribonucleotides. Three bases adenine, guanine,and cytosine are found in both DNA and RNA while thymine is normallyfound only in DNA and uracil is normally found only in RNA. Nucleotidescan have one, two or three attached phosphate groups and are sometimesreferred to as nucleoside phosphates. Nucleotides can contain modifiednucleosides having modified bases (e.g., 5-methyl cytosine) and modifiedsugar groups (e.g., 2′O-methyl ribosyl, 2′O-methoxyethyl ribosyl,2′fluoro ribosyl, 2′amino ribosyl, and the like). An example ofnon-natural bases that are used in the art are isocytidine andisoguanine.

A detectable label or a detectable molecule as used herein is intendedto mean an individual measurable moiety, such as a radioisotope,fluorochrome, dye, enzyme (including its effect on a substrate),nanoparticle, chemiluminescent marker, biotin, or other moiety known inthe art that is measurable by analytical methods. A detectable label ora detectable molecule can be attached to a nucleotide using methods wellknown in the art and exemplified herein.

Without limitations, examples of a detectable label or a detectablemolecule that can be utilized by some aspects described herein caninclude optical reporters or optical labels. Suitable optical reportersor optical labels include, but are not limited to, fluorescent reportersand chemiluminescent groups. A wide variety of fluorescent reporter dyesare known in the art. Typically, the fluorophore is an aromatic orheteroaromatic compound and can be a pyrene, anthracene, naphthalene,acridine, stilbene, indole, benzindole, oxazole, thiazole,benzothiazole, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluorescein, rhodamine or other like compound. Suitablefluorescent reporters include xanthene dyes, such as fluorescein orrhodamine dyes, including, but not limited to, fluorescent dyes soldunder the trade name Alexa Fluor® dyes (InvitrogenCorp.; Carlsbad,Calif.), fluorescein, fluorescein isothiocyanate (FITC), greenfluorescent dye sold under the trade name Oregon Green™, rhodamine,Texas red, tetrarhodamine isothiocynate (TRITC), 5-carboxyfluorescein(FAM), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX). Suitable fluorescent reporters also include the naphthylaminedyes that have an amino group in the alpha or beta position. Forexample, naphthylamino compounds include1-dimethylamino-naphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate,2-p-toluidinyl-6-naphthalene sulfonate, and5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Otherfluorescent reporter dyes include coumarins, such as3-phenyl-7-isocyanatocoumarin; acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p(2-benzoxazolyl)phenyl)maleimide; cyanines, such as Cy2,indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),indodicarbocyanine 5.5 (Cy5.5),3-(-carboxy-pentyl)-3′ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H, 15H-Xantheno [2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium,9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17octahydro-inner salt (TR or TexasRed); BODIPY™ dyes; benzoxadiazoles; stilbenes; pyrenes; and the like.Many suitable forms of these fluorescent compounds are available and canbe used.

Examples of fluorescent proteins suitable for use as imaging agentsinclude, but are not limited to, green fluorescent protein, redfluorescent protein (e.g., DsRed), yellow fluorescent protein, cyanfluorescent protein, blue fluorescent protein, and variants thereof(see, e.g., U.S. Pat. Nos. 6,403,374, 6,800,733, and 7,157,566).Specific examples of GFP variants include, but are not limited to,enhanced GFP (EGFP), destabilized EGFP, the GFP variants described inDoan et al, Mol. Microbiol, 55:1767-1781 (2005), the GFP variantdescribed in Crameri et al, Nat. Biotechnol., 14:315319 (1996), thecerulean fluorescent proteins described in Rizzo et al, Nat. Biotechnol,22:445 (2004) and Tsien, Annu. Rev. Biochem., 67:509 (1998), and theyellow fluorescent protein described in Nagal et al, Nat. Biotechnol.,20:87-90 (2002). DsRed variants are described in, e.g., Shaner et al,Nat. Biotechnol., 22:1567-1572 (2004), and include mStrawberry, mCherry,morange, mBanana, mHoneydew, and mTangerine. Additional DsRed variantsare described in, e.g., Wang et al, Proc. Natl. Acad. Sci. U.S.A.,101:16745-16749 (2004) and include mRaspberry and mPlum. Furtherexamples of DsRed variants include mRFPmars described in Fischer et al,FEBS Lett., 577:227-232 (2004) and mRFPruby described in Fischer et al,FEBS Lett, 580:2495-2502 (2006).

Amine-reactive and thiol-reactive fluorophores are available andgenerally used for labeling nucleotides and biomolecules. In someembodiments, nucleotides are fluorescently labeled during chemicalsynthesis, for example, incorporation of amines or thiols duringnucleotide synthesis permit addition of fluorophores. Fluorescentlylabeled nucleotides are commercially available. For example, uridine anddeoxyuridine triphosphates are available that are conjugated to tendifferent fluorophores that cover the spectrum.

In some embodiments, radioisotopes can be utilized as detectablemolecules or detectable molecules for labeling nucleotides including,for example, ³²P, ³³P, ³⁵S, ³H, and ¹²⁵I. These radioisotopes havedifferent half-lives, types of decay, and levels of energy which can betailored to match the needs of a particular experiment. For example, ³His a low energy emitter which results in low background levels, howeverthis low energy also results in long time periods for autoradiography.Radioactively labeled ribonucleotides and deoxyribonucleotides arecommercially available. Nucleotides are available that are radioactivelylabeled at the first, or α, phosphate group, or the third, or γ,phosphate group. For example, both [α-³²P]dATP and [γ-³²P]dATP arecommercially available. In addition, different specific activities forradioactively labeled nucleotides are also available commercially andcan be tailored for different experiments.

Suitable non-metallic isotopes include, but are not limited to, ¹¹C,¹⁴C, ¹³N, ¹⁸F, ¹²³I, ¹²⁴I, and ¹²⁵I. Suitable radioisotopes include, butare not limited to, ⁹⁹mTc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, Ga, ⁶⁸Ga, and ¹⁵³Gd.Suitable paramagnetic metal ions include, but are not limited to,Gd(III), Dy(III), Fe(III), and Mn(II). Suitable X-ray absorbers include,but are not limited to, Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au,Yb, Dy, Cu, Rh, Ag, and Ir. In some embodiments, the radionuclide isbound to a chelating agent or chelating agent-linker attached to theaggregate. Suitable radionuclides for direct conjugation include,without limitation, ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, and mixtures thereof.Suitable radionuclides for use with a chelating agent include, withoutlimitation, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In,¹¹⁷Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, andmixtures thereof. Suitable chelating agents include, but are not limitedto, DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA, their phosphonate analogs,and mixtures thereof. One of skill in the art will be familiar withmethods for attaching radionuclides, chelating agents, and chelatingagent-linkers to the particles.

Non-radioactive and non-fluorescent label monomers are also available.For example, biotin can be attached directly to nucleotides and detectedby specific and high affinity binding to avidin or streptavidin whichhas been chemically coupled to an enzyme catalyzing a colorimetricreaction (such as phosphatase, luciferase, or peroxidase). Digoxigeninlabeled nucleotides can also similarly be used for non-isotopicdetection of nucleic acids. Biotinylated and digoxigenin-labelednucleotides are commercially available.

In some embodiments, enzymatic reaction on a substrate can be utilized adetection method. In such embodiments, enzymes (e.g., horseradishperoxidase or alkaline phosphatase) can be linked to a nucleotide of anucleic acid label and/or decoder probe. During detection, a substrateon which the particular enzyme reacts can be added to induce acolorimetric reaction. Any enzyme-substrate reactions known in the artcan be used herein.

Very small particles, termed nanoparticles, also can be used asdetectable labels or detectable molecules to label decoder probes or anynucleic acids. These particles range from 1-1000 nm in size and includediverse chemical structures such as gold and silver particles andquantum dots.

When irradiated with angled incident white light, silver or goldnanoparticles ranging from 40-120 nm will scatter monochromatic lightwith high intensity. The wavelength of the scattered light is dependenton the size of the particle. Four to five different particles in closeproximity will each scatter monochromatic light which when superimposedwill give a specific, unique color. Alternatively, the goldnanoparticles can be detected or “developed” using a silver-baseddeveloper such that the tiny nanoparticle can be detected easily, evenwith naked eyes. The particles are being manufactured by companies suchas Genicon Sciences. Derivatized silver or gold particles can begenerally attached to a broad array of molecular molecules including,proteins, antibodies, small molecules, receptor ligands, and nucleicacids. For example, the surface of the particle can be chemicallyderivatized to allow attachment to a nucleotide, which can then beincorporated into a decoder probe.

Another type of nanoparticle that can be used as a detectable moleculeor detectable label are quantum dots. Quantum dots are fluorescingcrystals 1-5 nm in diameter that are excitable by a large range ofwavelengths of light. These crystals emit light, such as monochromaticlight, with a wavelength dependent on their chemical composition andsize. Quantum dots such as CdSe, ZnSe, InP, or InAs possess uniqueoptical properties.

Due to their very small size the quantum dots can be generally coupledinto oligonucleotides directly without affecting the solubility or useof the oligonucleotide. Thus, quantum dots can be coupled to decoderprobes as described herein. To synthesize a decoder probe-quantum dotcomplex by conventional batch chemistry, both the decoder probe and thequantum dot require at least a reactive group of different kinds thatcan be reacted with each other. For example, if a decoder probe has anamino group and a quantum dot has an aldehyde group, these groups canreact to form a Schiff base. A decoder probe can be derivatized toattach a single amino or other functional group using chemistry wellknown in the art. When a quantum dot is derivatized, the quantum dot canbe covered with a chemical reagent which results in coating the entiresurface of the nanoparticle with several functional groups.

A detectable molecule or detectable label can be attached to anucleotide of a decoder probe or nucleic acid using a variety of methodswell known in the art and described herein. For example, the detectablemolecule or detectable label can be directly attached to the nucleotidein a 1:1 correspondence by incorporation of a radioactive phosphate intothe phosphate backbone of the nucleotide. Also, for example, a generalmethod for labeling phosphates with a fluorescent label that employs animidazole derivative prepared from a BODIPY FL hydrazide has beenreported (Wang and Giese, Anal. Chem. 65: 3518 (1993).

Depending on the labeling moiety used, it can be desirable to derivatizeor chemically modify a nucleotide in order to bind the label monomer.These methods and chemistries are known in the art. In addition, alinker can be used to attach a detectable molecule or detectable labelto a nucleotide in a 1:1 correspondence. For example, a fluorescentlylabeled nucleotide such as fluorescein-12-dUTP can have a fluorophoremonomer attached via a four-atom aminoalkynyl group to the dUTPmolecule.

These nucleotides attached to detectable molecules or detectable labelscan be incorporated into a decoder probe or nucleic acid using severalmethods for labeling nucleic acids well known in the art. For example,enzymes such as DNA or RNA polymerases, Taq polymerases, terminaldeoxynucleotidyl transferases, or reverse transcriptases can be used toincorporate labeled nucleotides into decoder probes or nucleic acids.

Labeled nucleotides can be incorporated into decoder probe sequences ornucleic acids, for example, by nick translation. In this procedure DNAseI is used to create single-strand nicks in double stranded DNA and thenthe 5′ to 3′ exonuclease and 5′ to 3′ polymerase actions of E. coli DNApolymerase I are used to remove stretches of single stranded DNAstarting at the nicks and replace them with new strands made byincorporation of labeled nucleotides. Nick translation can utilize anylabeled nucleotide including radioactively labeled nucleotides andbiotinylated or digoxigenin labeled nucleotides. In a similar way T4 DNApolymerase can be used to incorporate labeled nucleotides. In addition,labeled nucleotides can be incorporated into nucleic acids using thepolymerase chain reaction (PCR) and Taq polymerases. The degree oflabeling can be controlled by including one, or up to all four labelednucleotides. In addition, the degree of labeling can be controlled byincreasing or decreasing the concentration of the labeled nucleotide(s).

Other methods for labeling decoder probes or nucleic acids includegenerating single-stranded cDNA from RNA by using a reversetranscriptase in the presence of labeled nucleotides. In addition, DNAcan be cloned into a vector with SP6 or T7 polymerase sites.Transcription in the presence of SP6 or T7 RNA polymerase and labelednucleotides results in a labeled RNA transcript. The transcript can belabeled to different degrees by including one or more labelednucleotides. In addition, several nucleotides within a nucleic acid canbe labeled, for example, by cloning DNA into a bacteriophage M13 basedvector. Then the Klenow fragment of DNA polymerase I and the M13universal probe primer can be used to synthesize the complementary standwith incorporation of labeled nucleotides.

Several methods are described above for incorporation of labelednucleotides into newly synthesized decoder probes or nucleic acids.Existing nucleic acids can also be labeled using several methods knownin the art. For example, RNA or DNA can be end-labeled with [γ-32P]ATPand T4 polynucleotide kinase. This kinase can be used to transfer theradioactive phosphate of ATP to a free 5′ OH group in either DNA or RNA.The enzyme also has a phosphatase activity and so two reactions arepossible. In the forward reaction, the enzyme catalyzes phosphorylationfollowing removal of 5′ terminal phosphates with alkaline phosphatase(or other phosphatase). In the exchange reaction, the kinase catalyzesthe exchange of an existing 5′ phosphate with the third or γ phosphateof ATP. The latter reaction is carried out in the presence of excess ATPand ADP for efficient phosphorylation. Using this method the radioactivephosphate of ATP is transferred to the end of the nucleic acid molecule.

Decoder probes or nucleic acids can also be labeled with terminaldeoxynucleotidyl transferase which adds labeled nucleotides onto the 3′end of DNA fragments. Both single and double-stranded DNAs aresubstrates for this enzyme. The large (Klenow) fragment of E. coli DNApolymerase I can also be used to label the ends of decoder probes ornucleic acids. Since this enzyme has a 5′ to 3′ polymerase activity itcan be used to “fill in” the 3′ ends of nucleic acid (e.g., DNA)fragments opposite of 5′ extensions or overhangs with labelednucleotides. End-labeling of decoder probes or nucleic acids usingpolynucleotide kinase or terminal deoxynucleotidyl transferase resultsin the incorporation of one detectable label or detectable molecule pernucleic acid. The “fill in” reaction can be used to label the decoderprobe or nucleic acid at one nucleotide per nucleic acid or at more thanone nucleotide per nucleic acid.

In addition, decoder probes or nucleic acids can be labeled bymodification of nucleotides within the nucleic acid sequences. Forexample, cytidine residues in DNA and RNA can be modified by reactionwith sodium bisulfite to form sulfonate intermediates that can then bedirectly coupled to hydrazides or aliphatic amines. Virtually any of thefluorescent, biotin or other hydrazides or aliphatic amines can be usedin this reaction. The bisulfite-activated cytidylic acid can also becoupled to aliphatic diamines such as ethylenediamine. Theamine-modified nucleic acids (e.g., DNA or RNA) can then be modifiedwith any of the amine-reactive dyes. In addition, phosphate groups canbe targeted in nucleic acids for labeling. Although phosphate groups ofnucleotides are not very reactive in aqueous solution, their terminalphosphate groups can react with carbodiimides and similar reagents incombination with nucleophiles to yield labeled phsophodiesters,phosphoramidates and phosphorothioates. For example, nucleic acids(e.g., DNA) can be reacted quantitatively with carbonyl diimidazole anda diamine such as ethylenediamine to yield a phosphoramidate that has afree primary amine and that this amine can then be modified withamino-reactive reagents. Fluorescent or biotinylated amines have beencoupled to the 5′ phosphate of tRNA using dithiodipyridine andtriphenylphosphine.

The bond between detectable molecules or detectable labels and decoderprobes or nucleic acids can be covalent bonds or non-covalent bonds thatare stable to hybridization. The detectable molecules or detectablelabels can be bound to a decoder probe or a nucleic acid in a sequencespecific manner, for example by the incorporation of a labelednucleotide into a sequence that has been digested by a restrictionenzyme. Alternatively the detectable molecules or detectable labels canbe bound to a decoder probe or a nucleic acid in a non-sequence specificmanner, for example by the incorporation of a label onto the terminalphosphate of a nucleic acid using [γ-³²P]ATP and T4 polynucleotidekinase.

Synthesis of Nucleic Acids (e.g., for at Least Part of the Nucleic AcidLabel Such as Pre-Determined Subsequences and/or Decoder Probe Sequences

The nucleic acids described herein can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between the labelattachment region and the annealed complementary polynucleotidesequences or segments, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the synthetic nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

Alternatively, the synthetic nucleic acid can be produced biologicallyusing a vector into which a nucleic acid has been subcloned. As oneexample, a linear single-stranded DNA backbone can be made from a doublestranded plasmid DNA using a four step protocol that includes (i)linearization of the dsDNA with a restriction enzyme, (ii)dephosphorylation with a thermolabile phosphatase, (iii) digestion witha second restriction enzyme to separate the cloning vector from thebackbone sequence, and (iv) digestion with a strand-specific lambdaexonuclease digestion, leaving only one strand of the backbone fragmentintact.

In various embodiments, the nucleic acids can be modified at the basemoiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup et al, 1996, Bioorganic &Medicinal Chemistry 40:5-23). As used herein, the terms “peptide nucleicacids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, inwhich the deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al, 1996, Bioorganic & MedicinalChemistry 4(1): 5-23; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci.USA 93: 14670-675.

In an exemplary embodiment, the selected novel nucleic acid sequence(e.g., DNA sequence) can be constructed synthetically as double-strandednucleic acid by a commercial gene synthesis company and cloned in anoriented fashion into a “phagemid”, a plasmid vector containing an M13or f1 phage intergenic (IG) region which contains the cis-actingsequences necessary for nucleic acid (e.g., DNA) replication and phageencapsidation, such as pUC119. The appropriate orientation of the clonedinsert relative to the phage origin of replication allows for thegeneration of a single-stranded DNA backbone which is the reversecomplement of the RNA molecules generated by in vitro transcription foreach label attachment region.

In order to generate the single-stranded nucleic acid (e.g., DNA)backbone (e.g., of at least part of the nucleic acid label and/ordecoder probes), the phagemid is transformed into an E. coli straincontaining an F′ episome. Subsequent infection of the transformedbacteria with a helper phage such as the M113 mutant K07 results in thesecretion of the phagemid carrying the single-stranded nucleic acidsequence, packaged phage from which the circular, single-strandednucleic acid is prepared using a standard protocol. This nucleic acid islinearized and the vector portion is excised by annealing short,complementary oligonucleotides to either end of the single-strandernucleic acid sequence to generate double-stranded restriction sites,followed by treatment with the appropriate restriction enzymes.

Analytes or Target Molecules

The terms “analyte,” “target analyte,” and “target molecule”, as usedinterchangeably herein, refer to the molecule detected, identified ormeasured by binding of a detection reagent described herein whose probereagent(s) recognize (i.e., are specific binding partners) thereto. Insome embodiments, a target molecule or an analyte can be, but is notlimited to, any of the following or any combinations of the following:nucleic acid, peptide, a polypeptide/protein (e.g., a bacterial or viralprotein or an antibody), a lipid, a carbohydrate, a glycoprotein, aglycolipid, a small molecule, an organic monomer, sugar, peptidoglycan,a cell, a virus or a drug. Nucleic acids that can be analyzed by themethods herein include: double-stranded DNA, single-stranded DNA,single-stranded DNA hairpins, DNA/RNA hybrids, RNA (e.g. mRNA or miRNA)and RNA hairpins. Generally, a target molecule can be a naturallyoccurring molecule or a cDNA of a naturally occurring molecule or thecomplement of said cDNA. In other embodiments, a target molecule can bemodified, e.g., by mutation or chemical reaction. In some embodiments, atarget molecule can be synthetic or recombinant.

In some embodiments, an analyte can be an analyte comprising at leastone post-translational modification, e.g., phosphorylations and/orglycosylations.

A target molecule or an analyte can be part of a sample that containsother components or can be the sole or major component of the sample. Atarget molecule or an analyte can be a component of a whole cell, tissueor body fluid, a cell or tissue extract, a fractionated lysate thereofor a substantially purified molecule. The target molecule can be presentin solution or attached to a solid substrate, including, for example, toa solid surface such as a chip, microarray, bead or a blotting membrane.Also the target molecule or analyte can have either a known or unknownstructure or sequence.

Sample

The methods, detection reagents and kits described herein can be used toanalyze a sample from any sources, e.g., but not limited to biologicalsamples (e.g., collected from organisms, animals or subjects),environmental samples, food, food byproduct, soil, archaeologicalsamples, extraterrestrial samples, or any combinations thereof.

In some embodiments, the term “sample” refers to a biological sample.The term “biological sample” as used herein denotes a sample taken orisolated from a biological organism, e.g., tissue cell culturesupernatant, cell lysate, a tissue sample (e.g., biopsy), a homogenateof a tissue sample from a subject or a fluid sample from a subject.Exemplary biological samples include, but are not limited to, blood,sputum, urine, cerebrospinal fluid, urine, sweat, mucus, nasaldischarge, vaginal fluids, spinal fluid, pleural fluid, nippleaspirates, lymph fluid, the external sections of the skin, respiratory,intestinal, and genitourinary tracts, tears, saliva, milk, feces, sperm,cells or cell cultures, serum, leukocyte fractions, smears, tissuesamples of all kinds, plants and parts of plants, microorganisms (suchas bacteria), viruses (such as cytomegalo virus, HIV, hepatitis B,hepatitis C, hepatitis 6 virus), yeasts, embryos, fungi, cell-freesample material, etc. The term also includes both a mixture of theabove-mentioned samples such as fungus-infected plants or whole humanblood containing mycobacteria as well as food samples that contain freeor bound nucleic acids, or proteins, or cells containing nucleic acidsor proteins, environmental samples which contain free or bound nucleicacids, or proteins, or cells containing nucleic acids or proteins. Theterm “biological sample” also includes untreated or pretreated (orpre-processed) biological samples.

A “biological sample” can contain cells from subject, but the term canalso refer to non-cellular biological material, such as non-cellularfractions of blood, saliva, or urine, that can be used to measure geneexpression levels. In some embodiments, the sample is from a resection,bronchoscopic biopsy, or core needle biopsy of a primary or metastatictumor, or a cell block from pleural fluid. In addition, fine needleaspirate samples can be used. Samples can be either paraffin-embedded orfrozen tissue. In some embodiments, a biological sample can comprise abiopsy, a surgically removed tissue, a swap, or any combinationsthereof.

The sample can be obtained by removing a sample of cells from a subject,but can also be accomplished by using previously isolated cells (e.g.isolated by another person). In addition, the biological sample can befreshly collected or a previously collected sample. Furthermore, thebiological sample can be utilized for the detection of the presenceand/or quantitative level of a biomolecule of interest. Representativetarget analytes include, but are not limited to nucleic acids, proteins,and derivatives and fragments thereof.

In some embodiments, the biological sample is an untreated biologicalsample. As used herein, the phrase “untreated biological sample” refersto a biological sample that has not had any prior sample pre-treatmentexcept for dilution and/or suspension in a solution. Exemplary methodsfor treating a biological sample include, but are not limited to,centrifugation, filtration, sonication, homogenization, heating,freezing and thawing, and any combinations thereof.

In accordance with some embodiments of various aspects described herein,a biological sample can be pre-processed, as described earlier, beforeemploying the detection reagents and the methods described herein. Insome embodiments, the biological sample can be filtered before isolatinga cellular material according to the methods, apparatus and kitsdescribed herein.

In some embodiments, the biological sample can be treated with achemical and/or biological reagent. Chemical and/or biological reagentscan be employed to protect and/or maintain the stability of the sampleor target analytes during processing. In addition, or alternatively,chemical and/or biological reagents can be employed to release or exposetarget analytes from other components of the sample. One exemplaryreagent is a protease inhibitor, which is generally used to protect ormaintain the stability of the target analytes during processing.

In some embodiments, the term “sample” as used herein can refer to anenvironmental sample (including, but not limited to, air, agricultural(e.g., but not limited to hydrofarms or hydroponic samples), pond,water, wastewater, and soil samples); biological warfare agent samples;research samples including extracellular fluids. In some embodiments, anenvironmental sample can comprise a sample collected from a workingsurface of an equipment or machine (e.g., but not limited to, food orpharmaceutical product processing equipment or machine), a device (e.g.,but not limited to, biomedical devices, implantation devices, fluiddelivery devices such as a tubing, and/or a catheter), and/or a buildingor dwellings (e.g., but not limited to, food processing plants,pharmaceutical manufacturing plants, hospitals, and/or clinics).

In some embodiments, a sample can comprise food (e.g., solid and/orfluid food as well as processed food) and/or food byproduct. Forexample, the methods, detection reagents and kits described herein canbe used to detect an analyte, e.g., a particular nutrient, in foodand/or food byproduct, e.g., but not limited to, meat, milk, yoghurt,bread, starch-based products, vegetables, and any combinations thereof.In some embodiments, the methods, detection reagents and kits describedherein can be used to detect a contaminant, e.g., bacteria, fungus,spores, molds, and/or viruses, in food and/or food byproduct.

In some embodiments, a sample can comprise a pharmaceutical product(e.g., but not limited to pills, tablets, gel capsules, syrups,vaccines, liquids, sprays, and any combinations thereof). For example,the methods, detection reagents and kits described herein can be used todetect the presence and/or measure the level of a particular activeagent present in a pharmaceutical product. In some embodiments, themethods, detection reagents and kits described herein can be used todetect a contaminant, e.g., bacteria, fungus, spores, molds, and/orviruses, in a pharmaceutical product.

In some embodiments, a sample can comprise an archaeological sample. Insome embodiments, an archaeological sample can be obtained or collectedfrom artifacts, architecture, biofacts (or ecofact, e.g., an objectfound at an archaeological site), cultural landscapes, and anycombinations thereof.

In some embodiments, a sample can comprise an extraterrestrial sample.For example, an extraterrestrial sample can be any object or specimen(e.g., rock, meteorite, and/or environmental samples) obtained orcollected from outer space or universe, and/or planets beyond the planetEarth, e.g., the moon, other planets (e.g., but not limited to, Mars,and/or Jupiter) and/or non-stellar objects.

Applications of the Detection Reagents

The detection reagents, compositions, methods and kits described hereincan be used for diagnostic, prognostic therapeutic and screeningpurposes. The inventions described herein provide the advantage thatmany different target analytes can be analyzed at one time from a singlesample using the methods described herein. This allows, for example, forseveral diagnostic tests to be performed on one sample.

When the probe reagent is a nucleic acid, the methods described hereincan discriminate between nucleotide sequences. The difference betweenthe target nucleotide sequences can be, for example, a single nucleicacid base difference, a nucleic acid deletion, a nucleic acid insertion,or rearrangement. Such sequence differences involving more than one basecan also be detected. In some embodiments, the oligonucleotide probesets have substantially the same length so that they hybridize to targetnucleotide sequences at substantially similar hybridization conditions.As a result, the process described herein is able to detect variouskinds of diseases or disorders, e.g., but not limited to, infectiousdiseases, genetic diseases, and cancer.

Without wishing to be bound, the detection reagents, compositions,methods and kits described herein can also be used for environmentalmonitoring, forensics, and food science. Examples of genetic analysesthat can be performed on nucleic acids include e.g., SNP detection, STRdetection, RNA expression analysis, promoter methylation, geneexpression, virus detection, viral subtyping and drug resistance.

In the area of environmental monitoring, some embodiments of variousaspects described herein can be used for detection, identification, andmonitoring of pathogenic and indigenous microorganisms in natural andengineered ecosystems and microcosms such as in municipal waste waterpurification systems and water reservoirs or in polluted areasundergoing bioremediation. It can also be used to detect plasmidscontaining genes that can metabolize xenobiotics, to monitor specifictarget microorganisms in population dynamic studies, or either todetect, identify, or monitor genetically modified microorganisms in theenvironment and in industrial plants.

Some embodiments of various aspects described herein can also be used ina variety of forensic areas, including for human identification formilitary personnel and criminal investigation, paternity testing andfamily relation analysis, HLA compatibility typing, and screening blood,sperm, or transplantation organs for contamination.

In the food and feed industry, some embodiments of various aspectsdescribed herein have a wide variety of applications. For example, itcan be used for identification and characterization of productionorganisms such as yeast for production of beer, wine, cheese, yogurt,bread, etc. Another area of use is related to the quality control andcertification of products and processes (e.g., livestock,pasteurization, and meat processing) for contaminants. Other usesinclude the characterization of plants, bulbs, and seeds for breedingpurposes, identification of the presence of plant-specific pathogens,and detection and identification of veterinary infections.

In some embodiments, the methods, detection reagents and kits describedherein can be used to detect the presence and/or measure the level of aparticular active agent present in a pharmaceutical product. In someembodiments, the methods, detection reagents and kits described hereincan be used to detect a contaminant, e.g., bacteria, fungus, spores,molds, and/or viruses, in a pharmaceutical product.

In some embodiments, the methods, detection reagents and kits describedherein can be used to detect the presence and/or measure the level of ananalyte of interest present in an archaeological sample, e.g., obtainedor collected from artifacts, architecture, biofacts (or ecofact, e.g.,an object found at an archaeological site), cultural landscapes, and anycombinations thereof.

In some embodiments, the methods, detection reagents and kits describedherein can be used to detect the presence and/or measure the level of ananalyte of interest present in an extraterrestrial sample, e.g., anyobject or specimen (e.g., rock, meteorite, and/or environmental samples)obtained or collected from outer space or universe, and/or planetsbeyond the planet Earth, e.g., the moon, other planets (e.g., but notlimited to, Mars, and/or Jupiter) and/or non-stellar objects. Forexample, the methods, detection reagents, and kits described herein canbe used to analyze the composition of an extratrerresterial sample orspecimen, e.g., a rock specimen, a water sample, and/or an air sample.

In some embodiments, the detection reagents and methods described hereincan be used to detect and/or identify analytes comprising apost-translation modification, e.g., phosphorylation or glycosylation.As such, in some embodiments, the detection reagents and methodsdescribed herein can differentiate between multiple differentglycosylation forms of recombinant therapeutic proteins

Immunohistochemistry: Some embodiments of various aspects describedherein can be used to perform antibody-based staining of cell or tissuesfor microscopic evaluation. This can involve either unfixed orunpermeabilized samples examined for surface or extracellular antigens,or permeabilized samples wherein intracellular antigens are also probed.While very common, immunohistochemistry is typically limited to probingwith a small set of antibodies at a time (due to the limitation ofavailable optical colors), and multiple staining cycles are generallyavoided, since the stripping of the preceding cycle's antibodies candamage the sample. Some embodiments of the detection reagents comprisingantibodies as probe reagents and nucleic acid labels can allow for manyantibodies to be used concurrently, which saves time and samplematerial, extract more data, and demand less prior knowledge of thesample. In some embodiments, without wishing to be bound by theory, whena subset of the probed antigens can overlap spatially, the detectionreagents with a plurality of contiguous pre-determined subsequences(e.g., each pre-determined subsequences contain one nucleotide, as shownin FIG. 1) can be used.

In-situ hybridization: Fluorescence in-situ hybridization (FISH) is atechnique for detecting (and/or quantifying) the presence of certaincellular DNA or RNA (often ribosomal RNA). As with otherfluorescence-based techniques, FISH is limited to the number of colorsavailable to the microscopy. Using some embodiments of the detectionreagents and/or methods described herein, the cells can be probed formany sequences simultaneously, thus allowing the user to save time andsample material, extract more data, and demand less prior knowledge ofthe sample.

Expression profiling: Nucleic-acid probe reagents of the detectionreagents can target messenger RNA or micro RNA and provide informationon the RNA-level expression state of the cell. Using some embodiments ofthe detection reagents and/or methods described herein, the assay cancapture and probe numerous mRNA and/or miRNA at once, reducing and/oreliminating potentially harmful stripping steps.

Western blots: Western blots are protein assays wherein proteins areseparated electrophoretically, transferred to a membrane and stained. Inmany cases, the staining is done using one or a few antibodies in orderto detect the corresponding antigens in the blot. By using someembodiments of the detection reagents and/or methods described herein,the blot can be simultaneously probed using a large number of antibodieswithout antibody-stripping steps. This can allow one blot to providesignificantly more information than the conventional Western blots whereone antibody is usually probed at a time and can require antibodystripping for the next antibody probing, thereby saving sample materialand time.

Diagnostic/Prognostic Methods: The present methods can be applied to theanalysis of biological samples obtained or derived from a patient so asto determine whether a diseased cell type is present in the sampleand/or to stage the disease.

In some embodiments, the methods described herein are used in thediagnosis of a condition. As used herein the term “diagnose” or“diagnosis” of a condition includes predicting or diagnosing thecondition, determining predisposition to the condition, monitoringtreatment of the condition, diagnosing a therapeutic response of thedisease, and prognosis of the condition, condition progression, andresponse to particular treatment of the condition. For example, a bloodsample can be assayed according to any of the methods described hereinto determine the presence and/or quantity of markers of a cancerous celltype in the sample, thereby diagnosing or staging the cancer.

In some embodiments, the detection reagents and methods described hereincan be directly used as contrast agents for in vivo or in situdiagnosis, in which detection reagents are administered to a subject,either by oral administration or local injection. The probe reagents ofthe detection reagents can bind to the target analytes, e.g., biomarkersfor specific diseases or disorders, and detection of the nucleic acidlabels using the methods described herein can then locate the targetanalytes. In some embodiments, the detection reagents and methodsdescribed herein can be used to locate microscopic tumors in a subject,where other conventional methods are not sensitive enough to detect suchmicroscopic tumors.

Cancers which can be detected by some embodiments of the processdescribed herein generally involve oncogenes, tumor suppressor genes, orgenes involved in DNA amplification, replication, recombination, orrepair. Examples of these include: BRCA1 gene, p53 gene, APC gene,Her2/Neu amplification, Bcr/Ab1, K-ras gene, and human papillomavirusTypes 16 and 18. Some embodiments of various aspects can be used toidentify amplifications, large deletions as well as point mutations andsmall deletions/insertions of the above genes in the following commonhuman cancers: leukemia, colon cancer, breast cancer, lung cancer,prostate cancer, brain tumors, central nervous system tumors, bladdertumors, melanomas, liver cancer, osteosarcoma and other bone cancers,testicular and ovarian carcinomas, head and neck tumors, and cervicalneoplasms.

Genetic diseases can also be detected by some embodiments of the processdescribed herein. This can be carried out by prenatal or post-natalscreening for chromosomal and genetic aberrations or for geneticdiseases. Examples of detectable genetic diseases include: 21hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, TurnerSyndrome, Duchenne Muscular Dystrophy, Down Syndrome or other trisomies,heart disease, single gene diseases, HLA typing, phenylketonuria, sicklecell anemia, Tay-Sachs Disease, thalassemia, Klinefelter Syndrome,Huntington Disease, autoimmune diseases, lipidosis, obesity defects,hemophilia, inborn errors of metabolism, and diabetes.

Alternatively, the methods described herein can be used to diagnosepathogen infections, for example infections by intracellular bacteriaand viruses, by determining the presence and/or quantity of markers ofbacterium or virus, respectively, in the sample.

A wide variety of infectious diseases can be detected by someembodiments of the process described herein. Typically, these are causedby bacterial, viral, parasite, and fungal infectious agents. Theresistance of various infectious agents to drugs can also be determinedusing some embodiments of various aspects described herein.

Bacterial infectious agents which can be detected by some embodiments ofvarious aspects described herein can include Escherichia coli,Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes,Mycobacterium tuberculosis, Mycobacterium aviumintracellulare, Yersinia,Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis,Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, B-Hemolyticstrep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia,Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza,Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacterpylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis,Rickettsial pathogens, Nocardia, and Acitnomycetes.

Fungal infectious agents which can be detected by some embodiments ofvarious aspects described herein can include Cryptococcus neoformans,Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis,Paracoccidioides brasiliensis, Candida albicans, Aspergillus fumigautus,Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, andMaduromycosis.

Viral infectious agents which can be detected by some embodiments ofvarious aspects described herein can include human immunodeficiencyvirus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g.,Hepatitis B Virus and Hepatitis C Virus), Epstein-Barr Virus,cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxoviruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses,toga viruses, bunya viruses, arena viruses, rubella viruses, and reoviruses.

Parasitic agents which can be detected by some embodiments of variousaspects described herein can include Plasmodium falciparum, Plasmodiummalaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus,Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica,Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli,Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascarislumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes,Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necatoramericanis.

In some embodiments, contacting a biological sample with the detectionreagents described herein immune-staining, in-situ hybridization or acombination, can be used for the purpose of detecting and identifyingpathogens in biological samples. In such embodiments, for example,surface or intracellular antigens, DNA, ribosomal RNA and/or messengerRNA can be detected with some embodiments of the detection reagents andmethods described herein. Detection reagents and methods describedherein can be especially useful in this context since a) which pathogenmay be in the sample is not known in advance, and b) each pathogen cellshould respond to one or few of the probes, facilitating the detectionreagent and nucleic acid label design.

Some embodiments of various aspects described herein can also be usedfor detection of drug resistance by infectious agents. For example,vancomycin-resistant Enterococcus faecium, methicillin-resistantStaphylococcus aureus, penicillin-resistant Streptococcus pneumoniae,multi-drug resistant Mycobacterium tuberculosis, and AZT-resistant humanimmunodeficiency virus can all be identified with some embodiments ofvarious aspects described herein.

Thus, the target molecules detected using the compositions and methodsdescribed herein can be either patient markers (such as a cancer marker)or markers of infection with a foreign agent, such as bacterial or viralmarkers.

Because of the quantitative nature of detection reagents, thecompositions and methods described herein can be used to quantitatetarget molecules whose abundance is indicative of a biological state ordisease condition, for example, blood markers that are upregulated ordownregulated as a result of a disease state.

In addition, the compositions and methods described herein can be usedto provide prognostic information that assists in determining a courseof treatment for a patient. For example, the amount of a particularmarker for a tumor can be accurately quantified from even a small samplefrom a patient. For certain diseases like breast cancer, overexpressionof certain genes, such as Her2-neu, indicate a more aggressive course oftreatment will be needed.

In some embodiments, the compositions and methods described herein canbe administered to a subject for in vivo diagnosis and/or monitoring ofa disease or disorder. Specific devices or methods known in the art forthe in vivo detection of fluorescence, e.g., from fluorophores orfluorescent proteins, include, but are not limited to, in vivonear-infrared fluorescence (see, e.g., Frangioni, Curr. Opin. Chem.Biol, 7:626-634 (2003)), the Maestro™ in vivo fluorescence imagingsystem (Cambridge Research & Instrumentation, Inc.; Woburn, Mass.), invivo fluorescence imaging using a flying-spot scanner (see, e.g.,Ramanujam et al, IEEE Transactions on Biomedical Engineering,48:1034-1041 (2001), and the like. Other methods or devices fordetecting an optical response include, without limitation, visualinspection, CCD cameras, video cameras, photographic film,laser-scanning devices, fluorometers, photodiodes, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microplate readers, or signal amplification usingphotomultiplier tubes.

Any device or method known in the art for detecting the radioactiveemissions of radionuclides in a subject is suitable for use in variousaspects described herein. For example, methods such as Single PhotonEmission Computerized Tomography (SPECT), which detects the radiationfrom a single photon gamma-emitting radionuclide using a rotating gammacamera, and radionuclide scintigraphy, which obtains an image or seriesof sequential images of the distribution of a radionuclide in tissues,organs, or body systems using a scintillation gamma camera, may be usedfor detecting the radiation emitted from a radiolabeled aggregate.Positron emission tomography (PET) is another suitable technique fordetecting radiation in a subject.

Analysis of Pathology Samples: RNA extracted from formaldehyde- orparaformaldehyde-fixed paraffin-embedded tissue samples is typicallypoor in quality (fragmented) and low in yield. This makes geneexpression analysis of low-expressing genes in histology samples orarchival pathology tissues extremely difficult and often completelyinfeasible. The detection reagents and methods described herein can fillthis unmet need by allowing the analysis of very small quantities oflow-quality total RNA.

To use detection reagents in such an application, total RNA can beextracted from formaldehyde- or paraformaldehyde-fixed paraffin-embeddedtissue samples (or similar) using commercially available kits such asRecoverAll Total Nucleic Acid Isolation Kit (Ambion) followingmanufacturer's protocols. RNA in such samples is frequently degraded tosmall fragments (200 to 500 nucleotides in length), and manyparaffin-embedded histology samples only yield tens of nanograms oftotal RNA. Small amounts (5 to 100 ng) of this fragmented total RNA canbe used directly as target analyte upon contact with the detectionreagents described herein, using the methods described herein describedherein.

Screening Methods: The methods described herein can be used, among otherthings, for determining the effect of a perturbation, including chemicalcompounds, mutations, temperature changes, growth hormones, growthfactors, disease, or a change in culture conditions, on various targetmolecules, thereby identifying target molecules whose presence, absenceor levels are indicative of particular biological states. In oneembodiment, some aspects described herein can be used to elucidate anddiscover components and pathways of disease states. For example, thecomparison of quantities of target molecules present in a disease tissuewith “normal” tissue allows the elucidation of important targetmolecules involved in the disease, thereby identifying targets for thediscovery/screening of new drug candidates that can be used to treatdisease.

Targeted Delivery Vehicles: In some embodiments, the therapeutic and/ordiagnostic agent can be encapsulated within the particles that provideconjugation sites for the probe reagents and nucleic acid labels. Insuch embodiments, the detection reagents and methods described hereincan be used to deliver a therapeutic and/or diagnostic agent to cellswhere the probe reagents of the detection reagents bind. Nucleic acidlabels of the detection reagents can be detected to monitor the locationof drug administration.

Cell Lineage Tracking: In some embodiments, the detection reagentsdescribed herein can be used to track cell lineage, e.g., in culture orin vivo. For example, the probe reagents of the detection reagents canbind to live cells of interest, allowing labeling and/or tracking ofindividual cell differentiation. Examples of such application include,but are not limited to, lineage tracking in stem cell differentiation,and lineage determination of dendritic cells and/or white blood cells.In some embodiments, these cells after lineage determination can befurther used accordingly.

Kits Comprising Detection Reagents

Kits for various assays are also provided herein. In some embodiments, akit can comprise: (a) a plurality of nucleic acid labels of thedetection reagents described herein; and (b) at least one couplingreagent required to conjugate the nucleic acid labels to probe reagentsof interest. In such embodiments, users can attach the provided nucleicacid labels to their probe reagents of interest to form their owndetection reagents described herein. Examples of the coupling reagentrequired for nucleic acid label-probe reagent conjugation can includeany reagents that are generally used to perform any of the conjugationmethods described herein, e.g., biotin and avidin-like molecules such asstreptavidin or neutravidin. However, in alternative embodiments, thekit can comprise a plurality of the detection reagents described hereinthat are ready for use and thus no nucleic acid label-probe reagentconjugation steps are required to be performed by users prior to use.

In some embodiments, the kit can further comprise at least one reagent,e.g., used in a readout method described herein. For example, if thereadout method is sequencing-based, the kit can further comprise atleast one agent used in sequencing-based readout. Alternatively, if thereadout method is hybridization-based, the kit can further comprise atleast one set of decoder probes complementary to at least a portion ofsubsequences of the detection reagents, wherein each subpopulation ofthe decoder probes comprises a different detectable label, eachdifferent detectable label producing a different signal signature.

Accordingly, in some embodiments, a kit for hybridization-based readoutcan comprise: (a) a plurality of nucleic acid labels of the detectionreagents described herein; (b) at least one reagent required toconjugate the nucleic acid labels to probe reagents of interest; and (c)at least one set of decoder probes complementary to at least a portionof subsequences of the detection reagents, wherein each subpopulation ofthe decoder probes comprises a different detectable label, eachdifferent detectable label producing a different signal signature. Insome embodiments, the kit can further comprise at least one reagent,e.g., used in the readout method.

In alternative embodiments, a kit for hybridization-based readout cancomprise: (a) a plurality of the detection reagents described herein;(b) at least one set of decoder probes complementary to at least aportion of subsequences of the detection reagents, wherein eachsubpopulation of the decoder probes comprises a different detectablelabel, each different detectable label producing a different signalsignature; and (c) at least one reagent.

In any embodiments of the kit described herein, the plurality of thedetection reagents can include one or more different kinds of thedetection reagents. In some embodiments, it is desirable to providedifferent kinds of the detection reagents (e.g., different types ofprobe reagents, target binding domains, and/or target analytes) inseparate containers. The number of different kinds of the detectionreagents provided in the kit can be tailored for each applicationdescribed herein. In some embodiments, the kit can comprise at least 2,at least 3, at least 4, at least 5, at least 6 at least 7, at least 8,at least 9, at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100 ormore different kinds of the detection reagents.

In any embodiments of the kit described herein, the kit can comprise aplurality of sets of decoder probes, e.g., e.g., 2, 3, 4, 5, 6, 7, 8, 9,10 or more sets of decoder probes, wherein each set of decoder probescan target a different pre-determined sequence of the nucleic acidlabel. In each set of the decoder probes, there can be about 2, 3, 4, 5,6, 7, 8, 9, 10 or more distinct populations of decoder probes. Thedecoder probes can be pre-labeled with at least one detectable label orunlabeled. In some embodiments where the decoder probes are notpre-labeled, the kit can further comprise one or more distinctdetectable labels provided in separate containers for labeling thedecoder probes. In some embodiments, the detectable label can be anoptical label described herein.

Examples of a reagent, e.g., used in a readout method, can include, butare not limited to, a readout reagent, a wash buffer, a signal removalbuffer, buffers for performing hybridization reactions, restrictionendonucleases, nucleic acid ligases, and any combinations thereof.

In some embodiments, the detection reagents can be provided in asolution phase. In other embodiments, the detection reagents can beimmobilized in a solid support or substrate.

By “substrate” or “solid support” or other grammatical equivalentsherein is meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association of beadsand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates is very large.Possible substrates include, but are not limited to, glass and modifiedor functionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, optical fiber bundles, and a variety of other polymers. Ingeneral, the substrates allow optical detection and do not themselvesappreciably fluoresce.

Generally the substrate is flat (planar), although as will beappreciated by those in the art, other configurations of substrates maybe used as well; for example, three dimensional configurations can beused, for example by embedding the detection reagents in a porous blockof plastic that allows sample access to the detection reagents and usinga confocal microscope for detection. In some embodiments, the detectionreagents can be placed on the inside surface of a tube, for flow-throughsample analysis to minimize sample volume. Preferred substrates includeoptical fiber bundles, and flat planar substrates such as glass,polystyrene and other plastics and acrylics. In some embodiment, thesolid support or substrate can be a multi-well plate.

In addition to the above mentioned components, the kit can includeinformational material. The informational material can be descriptive,instructional, marketing or other material that relates to the methodsdescribed herein and/or the use of the aggregates for the methodsdescribed herein. For example, the informational material describesmethods for administering the detection reagents to a subject, and/orincludes instructions to label decoder probes with detectable labelsprovided therein and/or instructions to conjugate at least one nucleicacid label to a probe reagent. The kit can also include a deliverydevice.

In some embodiments, the informational material can include instructionsto administer the formulation in a suitable manner, e.g., in a suitabledose, dosage form, or mode of administration (e.g., a dose, dosage form,or mode of administration described herein). In another embodiment, theinformational material can include instructions for identifying asuitable subject, e.g., a human, e.g., an adult human. The informationalmaterial of the kits is not limited in its form. In many cases, theinformational material, e.g., instructions, is provided in printedmatter, e.g., a printed text, drawing, and/or photograph, e.g., a labelor printed sheet. However, the informational material can also beprovided in other formats, such as Braille, computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is a link or contact information,e.g., a physical address, email address, hyperlink, website, ortelephone number, where a user of the kit can obtain substantiveinformation about the formulation and/or its use in the methodsdescribed herein. Of course, the informational material can also beprovided in any combination of formats.

In some embodiments, the kit contains separate containers, dividers orcompartments for each component and informational material. For example,each different component can be contained in a bottle, vial, or syringe,and the informational material can be contained in a plastic sleeve orpacket. In other embodiments, the separate elements of the kit arecontained within a single, undivided container. For example, theformulation is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label.

In some embodiments, the kit includes a plurality, e.g., a pack, ofindividual containers, each containing one or more unit dosage forms ofthe composition comprising the detection reagents. For example, the kitincludes a plurality of syringes, ampules, foil packets, or blisterpacks, each containing a single unit dose of the formulation. Thecontainers of the kits can be air tight and/or waterproof.

Embodiments of the Various Aspects Described Herein can be Illustratedby the Following Numbered Paragraphs.

1. A method for detecting a plurality of analytes in a sample,comprising:

a. contacting the sample with a composition comprising a plurality ofdetection reagents, wherein each subpopulation of the detection reagentstargets at least one different analyte, and wherein each detectionreagent comprises:

at least one probe reagent targeting an analyte and at least one nucleicacid label comprising one or a plurality of pre-determined subsequences,wherein said at least one probe reagent and said at least one nucleicacid label are conjugated together; and wherein at least a portion ofsaid one or the plurality of pre-determined subsequences form anidentifier of said at least one probe reagent; and

b. detecting in a temporally-sequential manner said one or the pluralityof the pre-determined subsequences of said detection reagent, whereinsaid detection of the subsequences each generates a signal signaturecorresponding to said subsequence, and wherein a temporal order of thesignal signatures corresponding to said one or the plurality of thesubsequences of said detection reagent identifies a subpopulation of thedetection reagents.

2. The method of paragraph 1, wherein said each subpopulation of thedetection reagents targets a set of analytes (e.g., at least twoanalytes or more).

3. The method of paragraph 1 or 2, wherein the temporal order of thesignal signatures corresponding to said one or the plurality of thesubsequences of said detection reagent is unique for each subpopulationof the detection reagents.

4. The method of any of paragraphs 1-3, wherein said detection reagentsare present in a soluble phase.

5. The method of any of paragraphs 1-4, further comprising processingsaid sample before said contacting with said plurality of detectionreagents.

6. The method of any of paragraphs 1-5, further comprising removing anyunbound detection reagents before the detecting step (b).

7. The method of any of paragraphs 1-6, further comprising comparingsaid temporal order of the signal signatures with different identifiersof said at least one probe reagent, wherein an agreement between thetemporal order of the signal signatures and a particular identifier ofsaid at least one probe reagent identifies the analyte in the sample.

8. The method of any of paragraphs 1-7, further comprising measuring theintensity of the signal signatures generated from each subpopulation ofthe detection reagents.

9. The method of paragraph 8, wherein the intensity of the signalsignatures generated from each subpopulation of the detection reagentsindicates an amount of the analyte.

10. The method of paragraph 8 or 9, wherein the intensity of the signalsignatures generated from each subpopulation of the detection reagentsis used in identification of the subpopulation of the detectionreagents.

11. The method of any of paragraphs 1-10, wherein said detecting of step(b) comprises sequencing.

12. The method of paragraph 11, wherein said sequencing is performed vialigation, hybridization, synthesis, amplification, or single-baseextension.

13. The method of any of paragraphs 1-12 wherein said detecting of step(b) comprises hybridizing a decoder probe with said subsequence, whereinsaid decoder probe comprises a detectable label.

14. The method of any of paragraphs 1-13, wherein said detecting of step(b) comprises:

a. hybridizing a set of decoder probes with a subsequence of thedetection reagents, wherein each subpopulation of the decoder probescomprises a detectable label, each detectable label producing a signalsignature;

b. detecting said signal signature produced by the hybridization of saidset of decoder probes;

c. optionally removing said different signal signature produced by thehybridization of said set of decoder probes; and

d. repeating steps (a) through (c) for other subsequences of saiddetection reagents, thereby producing a temporal order of the signalsignatures corresponding to said each detection reagent.

15. The method of paragraph 14, wherein said each subpopulation of thedecoder probes comprises a different detectable label, each differentdetectable label producing a different signal signature.

16. The method of paragraph 14 or 15, wherein said each subpopulation ofthe decoder probes is at least partially or completely complementary tosaid subsequence of the detection reagents.

17. The method of any of paragraphs 14-16, wherein at least two or moresubpopulations of the decoder probes are at least partially orcompletely complementary to the same subsequence of the detectionreagents.

18. The method of any of paragraphs 14-17, wherein said removing step isperformed by washing, heating, photobleaching, displacement, cleavage,enzymatic digestion, quenching, chemical degradation, bleaching,oxidation or any combinations thereof.

19. The method of any of paragraphs 14-18, wherein said detectable labelcomprises or is an optical label selected from the group consisting of asmall-molecule dye, a fluorescent molecule, a fluorescent protein, aquantum dot, Raman label, a chromophore, and any combinations thereof.

20. The method of any of paragraphs 14-19, wherein said detectable labelcomprises or is a colorimetric reagent.

21. The method of any of paragraphs 14-20, wherein said detectable labelcomprises or is a Raman label.

22. The method of any of paragraphs 1-21, wherein said signal signaturescomprise or are optical signatures.

23. The method of paragraph 22, wherein said optical signatures comprisesignatures of fluorescent color, visible light, no-color, Raman label,or any combinations thereof.

24. The method of paragraph 22, wherein said optical signatures comprisesignatures of one or more fluorescent colors, one or more visiblelights, one or more no-colors, one or more Raman labels, or anycombinations thereof.

25. The method of any of paragraphs 22-24, wherein said opticalsignatures are detected by optical imaging or spectroscopy.

26. The method of any of paragraphs 1-25, wherein said analytes areselected from the group consisting of antigens, receptors, proteins,peptides, sugars, glycoproteins, peptidoglycans, lipids, nucleic acids,oligonucleotides, cells, viruses, and any combinations thereof.

27. The method of any of paragraphs 1-26, wherein said nucleic acids areselected from the group consisting of cellular DNA or RNA, messengerRNA, microRNA, ribosomal RNA, and any combinations thereof.

28. The method of any of paragraphs 1-27, wherein said sample is aprotein sample immobilized on a solid support.

29. The method of paragraph 28, wherein the solid support is a blottingmembrane.

30. The method of any of paragraphs 1-27, wherein said sample is abiological sample.

31. The method of paragraph 30, wherein said biological sample comprisesone or more cells, one or more tissues, one or more fluids or anycombinations thereof.

32. The method of paragraph 30 or 31, wherein said biological samplecomprises blood, sputum, cerebrospinal fluid, urine, saliva, sperm,sweat, mucus, nasal discharge, vaginal fluids or any combinationsthereof.

33. The method of any of paragraphs 30-31, wherein the said biologicalsample comprises a biopsy, a surgically removed tissue, a swap or anycombinations thereof.

34. The method of paragraphs 1-27, wherein said sample comprises anenvironmental sample, food, food byproduct, soil, an archaeologicalsample, an extraterrestrial sample, or any combinations thereof.

35. The method of any of paragraphs 1-34, wherein said at least oneprobe reagent and said at least one nucleic acid label are conjugatedtogether by at least one linker.

36. The method of paragraph 35, wherein said linker is a bond.

37. The method of paragraph 35 or 36, wherein said linker is a linkermolecule.

38. The method of paragraph 37, wherein said linker molecule is apolymer, sugar, nucleic acid, peptide, protein, hydrocarbon, lipid,polyethylene glycol, crosslinker, or any combinations thereof.

39. The method of any of paragraphs 35-38, wherein said linker is aparticle.

40. The method of paragraph 39, wherein said particle is selected from agroup consisting of a gold nanoparticle, a magnetic bead ornanoparticle, a polystyrene bead, a nanotube, a nanowire, amicroparticle, and any combinations thereof.

41. The method of paragraph 40, wherein said particle is a nanoparticle.

42. The method of any of paragraphs 39-41, wherein said particle ismodified.

43. The method of any of paragraphs 39-42, wherein said particle iscoated with streptavidin or a derivative thereof.

44. The method of any of paragraphs 39-43, wherein said particle ismodified with at least one functional group.

45. The method of paragraph 44, wherein said at least one functionalgroup is selected from the group consisting of amine, carboxyl,hydroxyl, aldehyde, ketone, tosyl, silanol, chlorine, hydrazine,hydrazide, photoreactive groups, and any combinations thereof.

46. The method of any of paragraphs 35-45, wherein said linker ismultivalent.

47. The method of paragraph 46, wherein when the multivalent linker isan avidin-like molecule, both the probe reagent and the nucleic acidlabel are biotinylated.

48. The method of any of paragraphs 1-47, wherein said at least oneprobe reagent is selected from the group consisting of a nucleic acid,an antibody or a portion thereof, an antibody-like molecule, an enzyme,a cell, an antigen, a small molecule, a protein, a peptide, apeptidomimetic, a sugar, a carbohydrate, a lipid, a glycan, aglycoprotein, an aptamer, and any combinations thereof.

49. The method of any of paragraphs 1-48, wherein said at least oneprobe reagent is modified.

50. The method of any of paragraphs 1-49, wherein said at least oneprobe reagent is biotinylated.

51. The method of any of paragraphs 1-50, wherein said at least onenucleic acid label is single-stranded, double-stranded, partiallydouble-stranded, a hairpin, linear, circular, branched, a concatemer, orany combinations thereof.

52. The method of any of paragraphs 1-51, wherein said at least onenucleic acid label is modified.

53. The method of any of paragraphs 1-52, wherein said at least onenucleic acid label is designed for minimal cross-hybridization of baseswith each other.

54. The method of any of paragraphs 1-53, wherein said at least onenucleic acid label is conjugated to at least one detectable molecule.

55. The method of paragraph 54, wherein said at least one detectablemolecule is an optical molecule selected from the group consisting of asmall-molecule dye, a fluorescent protein, a quantum dot, a Raman label,a chromophore, and any combinations thereof.

56. The method of any of paragraphs 1-55, wherein each of said pluralityof the pre-determined subsequences comprises at least one base.

57. The method of any of paragraphs 1-56, wherein each of said pluralityof the pre-determined subsequences comprises from 1 to 100 nucleobases.

58. The method of any of paragraphs 1-57, wherein said plurality of thepre-determined subsequences are conjugated together by at least onesequence linker.

59. The method of paragraph 58, wherein said sequence linker is a bond.

60. The method of any of paragraphs 58-59, wherein said sequence linkeris a nucleotidic linker.

61. The method of paragraph 60, wherein said nucleotidic linker issingle-stranded, double-stranded, partially double-stranded, a hairpinor any combinations thereof.

62. The method of paragraph 60 or 61, wherein said nucleotidic linker isat least one nucleotide long.

63. The method of any of paragraphs 1-62, wherein said detection reagentcomprises one probe reagent and a plurality of nucleic acid labels.

64. The method of any of paragraphs 1-62, wherein said detection reagentcomprises a plurality of probe reagents and a nucleic acid label.

65. The method of any of paragraphs 1-62, wherein said detection reagentcomprises a plurality of probe reagents and a plurality of nucleic acidlabels.

66. The method of any of paragraphs 1-65, wherein the method is adaptedfor use in immunofluorescence.

67. The method of any of paragraphs 1-66, wherein the method is adaptedfor use in immunohistochemistry.

68. The method of any of paragraphs 1-67, wherein the method is adaptedfor use in fluorescence in situ hybridization.

69. The method of any of paragraphs 1-68, wherein the method is adaptedfor use in western blot.

70. A detection reagent comprising at least one probe reagent and atleast one nucleic acid label,

wherein said at least one nucleic acid label comprises at least onepre-determined subsequence to be detected in a temporally-sequentialmanner;

wherein said at least one pre-determined subsequence forms an identifierof said at least one probe reagent; and

wherein said at least one probe reagent and said at least one nucleicacid label are conjugated together.

71. The detection reagent of paragraph 70, wherein the detection reagentis present in a soluble phase.

72. The detection reagent of paragraph 70 or 71, wherein said at leastone probe reagent and said at least one nucleic acid label areconjugated together by at least one linker.

73. The detection reagent of paragraph 72, wherein said linker is abond.

74. The detection reagent of paragraph 72 or 73, wherein said linker isa linker molecule.

75. The detection reagent of paragraph 74, wherein said linker moleculeis a polymer, sugar, nucleic acid, peptide, protein, hydrocarbon, lipid,polyethelyne glycol, crosslinker or combination thereof.

76. The detection reagent of any of paragraphs 72-75, wherein saidlinker is a particle.

77. The detection reagent of paragraph 76, wherein said particle isselected from a group consisting of a gold nanoparticle, a magnetic beador nanoparticle, a polystyrene bead, a nanotube, a nanowire, amicroparticle, and any combinations thereof.

78. The detection reagent of paragraph 76 or 77, wherein said particleis a nanoparticle.

79. The detection reagent of any of paragraphs 76-78, wherein saidparticle is modified.

80. The detection reagent of any of paragraphs 76-79, wherein saidparticle is coated with streptavidin or a derivative thereof.

81. The detection reagent of any of paragraphs 76-80, wherein saidparticle is modified with at least one functional group.

82. The detection reagent of paragraph 81, wherein the at least onefunctional group is selected from the group consisting of amine,carboxyl, hydroxyl, aldehyde, ketone, tosyl, silanol, chlorine,hydrazine, hydrazide, photoreactive groups, and any combinationsthereof.

83. The detection reagent of any of paragraphs 72-82, wherein saidlinker is multivalent.

84. The detection reagent of paragraph 83, wherein when the multivalentlinker is an avidin-like molecule, both the probe reagent and thenucleic acid label are biotinylated.

85. The detection reagent of any of paragraphs 70-84, wherein said atleast one probe reagent is selected from the group consisting of anucleic acid, an antibody or a portion thereof, an antibody-likemolecule, an enzyme, a cell, an antigen, a small molecule, a protein, apeptide, a peptidomimetic, a sugar, a carbohydrate, a lipid, a glycan, aglycoprotein, an aptamer, and any combinations thereof.

86. The detection reagent of any of paragraphs 70-85, wherein said atleast one probe reagent is modified.

87. The detection reagent of any of paragraphs 70-86, wherein said atleast one probe reagent is biotinylated.

88. The detection reagent of any of paragraphs 70-87, wherein said atleast one nucleic acid label is single-stranded, double-stranded,partially double-stranded, a hairpin, linear, circular, branched, aconcatemer, or any combinations thereof.

89. The detection reagent of any of paragraphs 70-88, wherein said atleast one nucleic acid label is modified.

90. The detection reagent of any of paragraphs 70-89, wherein said atleast one nucleic acid label is designed for minimal cross-hybridizationof bases with each other.

91. The detection reagent of any of paragraphs 70-90, wherein said atleast one nucleic acid label is conjugated to at least one detectablemolecule.

92. The detection reagent of paragraph 91, wherein said at least onedetectable molecule is an optical molecule selected from the groupconsisting of small-molecule dye, a fluorescent protein, a quantum dot,a Raman label, a chromophore, and any combinations thereof.

93. The detection reagent of any of paragraphs 70-92, wherein said atleast one nucleic acid label comprises a plurality of pre-determinedsubsequences.

94. The detection reagent of any of paragraphs 70-93, wherein each ofsaid plurality of predetermined subsequences comprises at least onebase.

95. The detection reagent of any of paragraphs 70-94, wherein each ofsaid plurality of predetermined subsequences comprises from 1 to 100nucleobases.

96. The detection reagent of any of paragraphs 70-95, wherein saidplurality of the pre-determined subsequences are conjugated together byat least one sequence linker.

97. The detection reagent of paragraph 96, wherein said sequence linkeris a bond.

98. The detection reagent of any of paragraphs 96-97, wherein saidsequence linker is a nucleotidic linker.

99. The detection reagent of paragraph 98, wherein said nucleotidiclinker is single-stranded, double-stranded, partially double-stranded, ahairpin, or any combinations thereof.

100. The detection reagent of paragraph 98 or 99, wherein saidnucleotidic linker is at least one nucleotide long.

101. The detection reagent of any of paragraphs 70-100, wherein saiddetection reagent comprises one probe reagent and a plurality of nucleicacid labels.

102. The detecton reagent of any of paragraphs 70-101, wherein saiddetection reagent comprises a plurality of probe reagents and a nucleicacid label.

103. The detection reagent of any of paragraphs 70-102, wherein saiddetection reagent comprises a plurality of probe reagents and aplurality of nucleic acid labels.

104. The detection reagent of any of paragraphs 70-103, wherein thedetection reagent is adapted for use in immunofluorescence.

105. The detection reagent of any of paragraphs 70-104, wherein thedetection reagent is adapted for use in immunohistochemistry.

106. The detection reagent of any of paragraphs 70-105, wherein thedetection reagent is adapted for use in fluorescence in situhybridization.

107. The detection reagent of any of paragraphs 70-106, wherein thedetection reagent is adapted for use in western blot.

108. A kit comprising:

a. a plurality of the detection reagents of any of paragraphs 70-107;and

b. at least one reagent.

109. The kit of paragraph 108, wherein the kit further comprises atleast one set of decoder probes complementary to at least a portion ofsubsequences of the detection reagents, wherein each subpopulation ofthe decoder probes comprises a different detectable label, eachdifferent detectable label producing a different signal signature.

110. The kit of paragraph 108 or 109, wherein said detection reagentsare present in a soluble phase.

111. The kit of any of paragraphs 108-110, wherein said detectionreagents are immobilized in a multi-well plate.

112. The kit of any of paragraphs 108-111, wherein said at least onereagent is selected from the group consisting of a readout reagent, awash buffer, a signal removal buffer, and any combinations thereof.

113. A kit comprising:

a. a plurality of the nucleic acid labels of the detection reagents ofany of paragraphs 70-107;

b. at least one coupling agent that allows a user to conjugate thenucleic acid labels to the user's probe reagents of interest, therebyforming the detection reagents comprising the user's probe reagents ofinterest; and

c. at least one reagent.

114. The kit of paragraph 113, wherein the kit further comprises atleast one set of decoder probes complementary to at least a portion ofsubsequences of the detection reagents, wherein each subpopulation ofthe decoder probes comprises a different detectable label, eachdifferent detectable label producing a different signal signature.

115. The kit of paragraph 113 or 114, wherein the detection reagentscomprising the user's probe reagents of interest are present in asoluble phase.

116. The kit of any of paragraphs 113-115, wherein the detectionreagents comprising the user's probe reagents of interest areimmobilized in a multi-well plate.

117. The kit of any of paragraphs 113-116, wherein said at least onereagent is selected from the group consisting of a readout reagent, awash buffer, a signal removal buffer, and any combinations thereof.

Some Selected Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments of the aspects described herein, andare not intended to limit the claimed invention, because the scope ofthe invention is limited only by the claims. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not. Additionally, the term “comprising”or “comprises” includes “consisting essentially of” and “consisting of.”

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

As used herein, the term “identifier” generally refers to a uniqueexpression to distinguish variations from one to another among a classof substances, items, or objects. In particular embodiments, the term“identifier” as used herein refers to association of a uniquepre-determined subsequence to a specific probe reagent, thus conferringthe presence and identity of the probe reagent when the pre-determinedsubsequence is detected.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) above or below a reference level. The term refers to statisticalevidence that there is a difference. It is defined as the probability ofmaking a decision to reject the null hypothesis when the null hypothesisis actually true. The decision is often made using the p-value.

As used herein, the term “substantially” means a proportion of at leastabout 60%, or preferably at least about 70% or at least about 80%, or atleast about 90%, at least about 95%, at least about 97% or at leastabout 99% or more, or any integer between 70% and 100%. In someembodiments, the term “substantially” means a proportion of at leastabout 90%, at least about 95%, at least about 98%, at least about 99% ormore, or any integer between 90% and 100%. In some embodiments, the term“substantially” can include 100%.

The term “sphere” means a particle having an aspect ratio of at most3:1. The term “aspect ratio” means the ratio of the longest axis of anobject to the shortest axis of the object, where the axes are notnecessarily perpendicular.

The term “rod” means a particle having a longest dimension of at most5000 nm, and having an aspect ratio of from 3:1 to 20:1.

The term “prism” means a particle having at least two non-parallel facesconnected by a common edge.

As used herein, the term “pharmaceutically acceptable carrier” refers toa pharmaceutically-acceptable material, composition or vehicle foradministration of the detection reagents. Pharmaceutically acceptablecarriers include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like which are compatible with the activity of thedetection reagents and are physiologically acceptable to the subject.Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alchols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” are used interchangeably herein.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “therapeutic agent” refers to a biological orchemical agent used for treatment, curing, mitigating, or preventingdeleterious conditions in a subject. The term “therapeutic agent” alsoincludes substances and agents for combating a disease, condition, ordisorder of a subject, and includes drugs, diagnostics, andinstrumentation. “Therapeutic agent” also includes anything used inmedical diagnosis, or in restoring, correcting, or modifyingphysiological functions. The terms “therapeutic agent” and“pharmaceutically active agent” are used interchangeably herein.

A therapeutic agent can be selected according to the treatment objectiveand biological action desired. Thus, a therapeutic agent can be selectedfrom any class suitable for the therapeutic objective. Further, thetherapeutic agent may be selected or arranged to provide therapeuticactivity over a period of time.

Exemplary pharmaceutically active compound include, but are not limitedto, those found in Harrison's Principles of Internal Medicine, 13thEdition, Eds. T. R. Harrison McGraw-Hill N.Y., N.Y.; Physicians DeskReference, 50th Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman,1990; United States Pharmacopeia, The National Formulary, USP XII NFXVII, 1990; current edition of Goodman and Oilman's The PharmacologicalBasis of Therapeutics; and current edition of The Merck Index, thecomplete content of all of which are herein incorporated in itsentirety.

Exemplary pharmaceutically active agents include, but are not limitedto, steroids and nonsteroidal anti-inflammatory agents, antirestenoticdrugs, antimicrobial agents, angiogenic factors, calcium channelblockers, thrombolytic agents, antihypertensive agents, anti-coagulants,antiarrhythmic agents, cardiac glycosides, and the like.

In some embodiments, the therapeutic agent is selected from the groupconsisting of salicylic acid and derivatives (aspirin), para-aminophenoland derivatives (acetaminophen), arylpropionic acids (ibuprofen),corticosteroids, histamine receptor antagonists and bradykinin receptorantagonists, leukotriene receptor antagonists, prostaglandin receptorantagonists, platelet activating factor receptor antagonists,sulfonamides, trimethoprim-sulfamethoxazole, quinolones, penicillins,cephalosporin, basic fibroblast growth factor (FGF), acidic fibroblastgrowth factor, vascular endothelial growth factor, angiogenictransforming growth factor alpha and beta, tumor necrosis factor,angiopoietin, platelet-derived growth factor, dihydropyridines (e.g.,nifedipine, benzothiazepines such as dilitazem, and phenylalkylaminessuch as verapamil), urokinase plasminogen activator, urokinase,streptokinase, angiotensin converting enzyme (ACE) inhibitors,spironolactone, tissue plasminogen activator (tPA), diuretics,thiazides, antiadrenergic agents, clonidine, propanolol,angiotensin-converting enzyme inhibitors, captopril, angiotensinreceptor antagonists, losartan, calcium channel antagonists, nifedine,heparin, warfarin, hirudin, tick anti-coagulant peptide, and lowmolecular weight heparins such as enoxaparin, lidocaine, procainamide,encainide, flecanide, beta adrenergic blockers, propranolol, amiodarone,verpamil, diltiazem, nickel chloride, cardiac glycosides, angiotensinconverting enzyme inhibitors, angiotensin receptor antagonists,nitrovasodilators, hypolipidemic agents (e.g., nicotinic acid, probucol,etc.), bile acid-binding resins (e.g., cholestyramine, and fibric acidderivatives e.g., clofibrate), HMG CoA reductase inhibitors, HMG CoAsynthase inhibitors, squalene synthase inhibitors, squalene epoxidaseinhibitors, statins (e.g., lovastatin, cerivastatin, fluvastatin,pravastatin, simvaststin, etc.), anti-psychotics, SSRIs, antiseizuremedication, contraceptives, systemic and local analgesics (chronic pain,bone growth/remodeling factors (osteoblast/osteoclast recruiting andstimulating factors), neurotransmitters (L-DOPA, Dopamine,neuropeptides), emphysema drugs, TGF-beta), rapamycin, naloxone,paclitaxel, amphotericin, Dexamethasone, flutamide, vancomycin,phenobarbital, cimetidine, atenolol, aminoglycosides, hormones (e.g.,thyrotropin-releasing hormone, p-nitrophenyl beta-cellopentaosideandluteinizing hormone-releasing hormone), vincristine, amiloride, digoxin,morphine, procainamide, quinidine, quinine, ranitidine, triamterene,trimethoprim, vancomycin, aminoglycosides, and penicillin, andpharmaceutically acceptable salts thereof.

As used herein, the term “fluorescent color” refers to a color emittedby any fluorophore (e.g., fluorescent dye, fluorescent particles such asquantum dots, and/or fluorescent proteins) upon light excitation and/orelectromagnetic radiation. Fluorescent dye as disclosed herein refers toa dye which exhibits energy and is visible under illumination at apredefined wavelength. Numerous fluorescent molecules are commerciallyavailable and can be adapted for use in the methods, detection reagentsand kits as disclosed herein, and include those from Molecular Probes,Sigma and similar other commercial sources. In some embodiments, a“fluorescent color” can be produced by phosphorescence. In someembodiments, a “fluorescent color” can be produced by luminescence(including bioluminescence).

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated may be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

EXAMPLES Example 1: Exemplary Hybridization-Based Detection Methods ofDetection Reagents

A solution suspension of streptavidin-coated Dynabeads (of about 1 μm),each conjugated to one of six nucleic acid labels and a correspondingprobe reagent specific for a target analyte, wherein each of the nucleicacid labels comprised at least one pre-determined subsequence, e.g., afirst (e.g., A1, A2, and A3) and a second (B1, B2, and B3)pre-determined subsequence, was prepared. The nucleic acid labels cancomprise a nucleic acid sequence of any length. In some embodiments, thenucleic acid labels can comprise at least 15 nucleotides, at least 20nucleotides, at least 22 nucleotides, or at least 24 nucleotides. Asample containing multiple target analytes was contacted with the targetanalyte-specific Dynabeads, so that the Dynabeads would bind to therespective target analyte on the sample. Any unbound Dynabeads was thenremoved, e.g., by washing. In the first readout, a set of three decoderprobes, each with a distinct sequence complementary to the firstpre-determined subsequences (e.g., A1*, A2*, and A3*) and acorresponding optical label (e.g., selected from red, green or blank),was added to the sample with bound Dynabeads, and then imaged withfluorescent microscopy (FIG. 6A). The fluorescent signal was thenremoved, e.g., by photobleaching, from the previous stage before thenext readout. In the second readout, a different set of three decoderprobes, each with a distinct complementary sequence to the secondpre-determined subsequences (e.g., B1*, B2*, B3*) and a correspondingoptical label (e.g., selected from red, green blank), was added to thephotobleached sample, and then imaged with fluorescent microscopy (FIG.6B). The temporal sequence of the optical signatures obtained from eachbead can then be analyzed to determine the identity of the probe andthus the presence of the corresponding target analyte on the sample.Based on the intensity and/or coverage of the optical signals, theamount of the target analyte can also be determined.

By way of example only, a biotinylated anti-C. albicans antibody (e.g.,an commercially-available antibody from Pierce Antibodies PA1-27145) wasconjugated with a plurality of different biotinylated nucleic acidlabels (e.g., 8 different biotinylated SeqTag label sequences) using anyconjugation methods known in the art. In one embodiment, thebiotinylated anti-C. albicans antibody was conjugated with a pluralityof different biotinylated nucleic acid labels (e.g., biotinylated SeqTaglabel sequences) using a streptavidin-like protein (e.g., Neutravidin)as a bridge. The SeqTag label sequences are shown in Table 5. The SeqTaglabel sequences can comprise at least about 24 nucleotides. In someembodiments, the SeqTag label sequences can comprise DNA, RNA or acombination thereof. Each conjugate was incubated with a samplecomprising C. albicans, washed, and readout using any readout method asdescribed herein, e.g., the displacement-hybridization method.

TABLE 5 DNA sequences of exemplary SeqTag labels SEQ ID SeqTag NO: labelDNA Sequence 59 SeqTag 1 5′-Biotin-TTTCGCTTTCTGTAATGGAGTGGA-3′ 60SeqTag 2 5′-Biotin-TTTCGCTTTAGCCTAAGTGAAATC-3′ 61 SeqTag 35′-Biotin-TTTCGCTTTTTTGGGGAAAAGACA-3′ 62 SeqTag 45′-Biotin-TTTCGCTTTTAGGCATTAGCATTG-3′ 63 SeqTag 55′-Biotin-TTTCGCTTTGGAAGCACCTATTCC-3′ 64 SeqTag 65′-Biotin-TTTCGCTTTCTGGAGAAAGGGCCA-3′ 65 SeqTag 75′-Biotin-TTTCGCTTTCGGTTCCAAAGACAC-3′ 66 SeqTag 85′-Biotin-TTTCGCTTTGAAGCCGGTTATAGC-3′

During the displacement hybridization method, by way of example only, asshown in FIG. 7, the yeast was incubated in readout step 1 with amixture of all three “set 1” decoder probes, followed by an incubationwith a mixture of the “set 2” decoder probes and the “set 1 displacers”(which are nucleic acid sequences to remove “set 1” fluorescence), andsimilarly with appropriate sets of decoder probes and displacers forsteps 3 and 4. The decoder probes can comprise a nucleic acid sequence(e.g., DNA, RNA or a combination thereof) and a detection label, e.g.,at its 5′ end. The nucleic acid sequence of the decoder probes can be ofany length. In some embodiments, the decoder probes can comprise atleast about 10 nucleotides, at least about 12 nucleotides, at leastabout 14 nucleotides, at least about 16 nucleotides, at least about 18nucleotides, at least about 20 nucleotides or longer. Detection labelscan be any art-recognized label described herein, e.g., fluorescentdetection labels such as FAM, Cy3, and C5, or any labels describedherein. Probe displacers, as described earlier, are nucleic acidsequences that are used to remove or displace the previous decoderprobes hybridized to the nucleic acid labels (e.g., SeqTag labels) suchthat a different set of decoder probes can be added and hybridized tothe nucleic acid labels. The probe displacers can have a nucleic acidsequence of any length. In some embodiments, the nucleic acid sequenceof the probe displacers can be longer than that of the decoder probes.In some embodiments, the probe displacers can comprise at least about 12nucleotides, at least about 14 nucleotides, at least about 16nucleotides, at least about 18 nucleotides, at least about 20nucleotides, at least about 21 nucleotides or longer. Tables 2 and 3show DNA sequences of exemplary decoder probes (or readout probes) andprobe displacers, respectively. As seen in FIG. 7, each biotinylatedanti-C albicans antibody conjugated with a different SeqTag labelsequence properly stained the yeast and fluorescenced only as designatedby its SeqTag label with appropriate sets of decoder probes and probedisplacer in each readout step.

TABLE 6 DNA sequences of exemplary decoder probes SEQ ID Readout NO:probe DNA Sequence 67 Set 1 FAM 5′-FAM-TTTTCCACTCCATTACAG-3′ 68Set 1 Cy3 5′-Cy3-TTTGATTTCACTTAGGCT-3′ 69 Set 1 Cy55′-Cy5-TTTTGTCTTTTCCCCAAA-3′ 70 Set 2 FAM 5′-FAM-TTTCAATGCTAATGCCTA-3′71 Set 2 Cy3 5′-Cy3-TTTGGAATAGGTGCTTCC-3′ 72 Set 2 Cy55′-Cy5-TTTTGGCCCTTTCTCCAG-3′ 73 Set 3 FAM 5′-FAM-TTTGTGTCTTTGGAACCG-3′74 Set 3 Cy3 5′-Cy3-TTTGCTATAACCGGCTTC-3′

TABLE 7 DNA sequences of exemplary probe displacers SEQ ID Probe NO:Displacer DNA Sequence 75 Set 1 FAM- 5'-TCCACTCCATTACAGAAAGCG-3′Displacer 76 Set 1 Cy3- 5'-GATTTCACTTAGGCTAAAGCG-3′ Displacer 77Set 1 Cy5- 5'-TGTCTTTTCCCCAAAAAAGCG-3′ Displacer 78 Set 2 FAM-5'-CAATGCTAATGCCTAAAAGCG-3′ Displacer 79 Set 2 Cy3-5'-GGAATAGGTGCTTCCAAAGCG-3′ Displacer 80 Set 2 Cy5-5'-TGGCCCTTTCTCCAGAAAGCG-3′ Displacer 81 Set 3 FAM-5'-TGTGTCTTTGGAACCGAAAGCG-3′ Displacer 82 Set 3 Cy3-5'-GCTATAACCGGCTTCAAAGCG-3′ Displacer

Content of all patents and other publications identified herein isexpressly incorporated herein by reference for all purposes. Thesepublications are provided solely for their disclosure prior to thefiling date of the present application. Nothing in this regard should beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention or for any otherreason. All statements as to the date or representation as to thecontents of these documents is based on the information available to theapplicants and does not constitute any admission as to the correctnessof the dates or contents of these documents.

What is claimed is:
 1. A method for identifying an analyte, comprising:(a) contacting a cell or tissue sample comprising said analyte with adetection reagent comprising (i) a probe targeting said analyte and (ii)a nucleic acid label coupled to said probe, to permit said probe to bindto said analyte, wherein said nucleic acid label comprises a pluralityof subsequences; (b) generating a set of signal signatures in said cellor tissue sample at least in part by (i) coupling a first decoder probeto a first subsequence of said plurality of subsequences, wherein saidfirst decoder probe comprises a first detectable label, (ii) detecting afirst signal signature from said first detectable label, (iii) couplinga second decoder probe to a second subsequence of said plurality ofsubsequences, wherein said second decoder probe comprises a seconddetectable label, and (iv) detecting a second signal signature from saidsecond detectable label; and (c) processing said set of signalsignatures to identify said analyte.
 2. The method of claim 1, whereinsaid analyte is selected from the group consisting of an antigen, aprotein, a peptide, a sugar, a glycoprotein, a peptidoglycan, a lipid, anucleic acid, a virus, and any combination thereof.
 3. The method ofclaim 1, wherein said cell or tissue sample is immobilized on a solidsubstrate or support.
 4. The method of claim 1, wherein said probe isselected from the group consisting of a nucleic acid, an antibody, anantigen binding fragment of an antibody, an antibody-like molecule, anenzyme, an antigen, a small molecule, a protein, a peptide, apeptidomimetic, a sugar, a carbohydrate, a lipid, a glycan, aglycoprotein, an aptamer, and any combination thereof.
 5. The method ofclaim 1, wherein said probe and said nucleic acid label are conjugatedtogether by at least one linker.
 6. The method of claim 5, wherein saidat least one linker is a particle.
 7. The method of claim 1, whereinsaid first subsequence and said second subsequence of said plurality ofsubsequences are conjugated together by a nucleotidic linker.
 8. Themethod of claim 1, wherein said probe is a nucleic acid probe andwherein said probe and said nucleic acid label are conjugate together bya nucleotidic linker.
 9. The method of claim 8, wherein said nucleicacid probe and said nucleic acid label are single-stranded.
 10. Themethod of claim 1, wherein said probe is a nucleic acid probe andwherein said probe and said nucleic acid label are conjugate together bya phosphodiester bond.
 11. The method of claim 1, wherein said probecomprises a plurality of nucleic acid labels, which plurality of nucleicacid labels comprises said nucleic acid label.
 12. The method of claim11, wherein said plurality of nucleic acid labels comprise identicalnucleic acid sequences.
 13. The method of claim 1, wherein said firstdetectable label or said second detectable label comprises an opticallabel.
 14. The method of claim 13, wherein said optical label comprisesa small molecule dye, a fluorescent molecule, a quantum dot, achromophore, a chromogenic molecule, a Raman label, or any combinationthereof.
 15. The method of claim 1, wherein said analyte is a proteinand said probe is an antibody or an antigen-binding fragment of anantibody that binds said protein.
 16. The method of claim 1, whereinsaid probe is an aptamer that binds said analyte.
 17. The method ofclaim 1, further comprising, prior to coupling said second decoder probeto said second subsequence, removing said first signal signature. 18.The method of claim 17, wherein removing said first signal signaturecomprises washing, heating, photo-bleaching, displacement, cleavage,enzymatic digestion, quenching, chemical degradation, bleaching,oxidation, or any combination thereof.
 19. The method of claim 17,wherein removing said first signal signature comprises removing saidfirst decoder probe.
 20. The method of claim 19, wherein removing saidfirst decoder probe comprises using a buffer comprising a detergent or adenaturant.
 21. The method of claim 1, wherein said plurality ofsubsequences form an identifier of said probe and wherein (c) comprisescomparing said set of signal signatures with said identifier, wherein anagreement between said set of signal signatures and said identifieridentifiers said analyte.
 22. The method of claim 1, wherein saidanalyte is a ribonucleic acid (RNA) molecule and wherein said probecomprises a nucleic acid sequence that hybridizes to said RNA molecule.23. The method of claim 1, wherein said first detectable label or saidsecond detectable label comprises a fluorescent label.
 24. The method ofclaim 1, wherein said first subsequence and said second subsequence areeach 3-50 nucleotides in length.
 25. The method of claim 24, whereinsaid first subsequence and said second subsequence are each 5-30nucleotides in length.
 26. The method of claim 1, further comprising,prior to (b), binding an additional detection reagent to said analyte,wherein said additional detection reagent comprises (i) an additionalprobe and (ii) an additional nucleic acid label comprising an additionalplurality of subsequences; and wherein generating said set of signalsignatures in said cell or tissue sample further comprises (i) couplinga third decoder prior to a subsequence of said additional plurality ofsubsequences, wherein said third decoder probe comprises a thirddetectable label, (ii) detecting a third signal signature from saidthird detectable label, (iii) coupling a fourth decoder probe to anadditional subsequence of said additional plurality of subsequences,wherein said fourth decoder probe comprises a fourth detectable label,and (iv) detecting a fourth signal signature from said fourth detectablelabel.
 27. A method for identifying an analyte, comprising: (a)contacting a cell or tissue sample with a nucleic acid detection reagentcomprising (i) a probe sequence targeting a ribonucleic acid (RNA)molecule and (ii) a label sequence comprising a plurality ofsubsequences that form an identifier of said probe; generating a set ofsignal signatures in said cell or tissue sample at least in part by (i)coupling a first decoder probe to a first subsequence of said pluralityof subsequences, wherein said first decoder probe comprises a firstfluorescent label, (ii) detecting a first signal signature from saidfirst fluorescent label, wherein said first signal signature isassociated with said first subsequence (iii) coupling a second decoderprobe to a second subsequence of said plurality of subsequences, whereinsaid second decoder probe comprises a second fluorescent label, and (iv)detecting a second signal signature from said second fluorescent label,wherein said second signal signature is associated with said secondsubsequence; and (c) comparing said set of signal signatures with saididentifier to identify said RNA molecule.
 28. The method of claim 27,wherein said first subsequence and said second subsequence are each 3-50nucleotides in length.
 29. The method of claim 28, wherein said firstsubsequence and said second subsequence are each 5-30 nucleotides inlength.